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WO2000073498A1 - Compositions et procede pour le traitement et le diagnostic de troubles de l'immunite - Google Patents

Compositions et procede pour le traitement et le diagnostic de troubles de l'immunite Download PDF

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WO2000073498A1
WO2000073498A1 PCT/US2000/014986 US0014986W WO0073498A1 WO 2000073498 A1 WO2000073498 A1 WO 2000073498A1 US 0014986 W US0014986 W US 0014986W WO 0073498 A1 WO0073498 A1 WO 0073498A1
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gene
cell
cells
gene product
genes
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Douglas A. Levinson
Clare M. Lloyd
Sean A. Mccarthy
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/08—Antibacterial agents for leprosy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to methods and compositions for the treatment and diagnosis of immune disorders, especially T lymphocyte-related disorders, including, but not limited to, chronic inflammatory diseases and disorders, such as Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ- specific autoimmunity, including multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g.
  • genes which are differentially expressed within and among T helper (TH) cells and TH cell subpopulations which include, but are not limited to TH0 , TH1 and TH2 cell subpopulations are identified. Genes are also identified via the ability of their gene products to interact with gene products involved in the differentiation, maintenance and effector function of such TH cells and TH cell subpopulations. Among the genes identified are ones involved in repair or recovery of tissue from ischemic disorders or injuries. The genes identified can be used diagnostically or as targets for therapeutic intervention.
  • the present invention provides methods for the identification and therapeutic use of compounds as treatments of immune disorders, especially TH cell subpopulation-related disorders.
  • the present invention also provides methods for treating ischemic disorders or injuries. Additionally, methods are provided for the diagnostic evaluation and prognosis of TH cell subpopulation-related disorders, for the identification of subjects exhibiting a predisposition to such conditions, for monitoring patients undergoing clinical evaluation for the treatment of such disorders, and for monitoring the efficacy of compounds used in clinical trials.
  • CTLs cytotoxic T lymphocytes
  • TH cells helper T lymphocytes
  • CTLs recognize and kill cells which display foreign antigens of their surfaces.
  • CTL precursors display T cell receptors that recognize processed peptides derived from foreign proteins, in conjunction with class I MHC molecules, on other cell surfaces. This recognition process triggers the activation, maturation and proliferation of the precursor CTLs, resulting in CTL clones capable of destroying the cells exhibiting the antigens recognized as foreign.
  • TH cells are involved in both humoral and cell-mediated forms of effector immune responses.
  • humoral, or antibody, immune response antibodies are produced by B lymphocytes through interactions with TH cells.
  • extracellular antigens are endocytosed by antigen-presenting cells (APCs) , processed, and presented preferentially in association with class II major histocompatibility complex (MHC) molecules to CD4 + class II MHC-restricted TH cells.
  • APCs antigen-presenting cells
  • MHC major histocompatibility complex
  • the cell-mediated, or cellular, immune response functions to neutralize microbes which inhabit intracellular locations.
  • Foreign antigens such as, for example, viral antigens, are synthesized within infected cells and presented on the surfaces of such cells in association with class I MHC molecules. This, then, leads to the stimulation of the CD8 + class I MHC-restricted CTLs.
  • agents such as ycobacteria, which cause tuberculosis and leprosy, are engulfed by macrophages and processed in vacuoles containing proteolytic enzymes and other toxic substances. While these macrophage components are capable of killing and digesting most microbes, agents such as mycobacteria survive and multiply.
  • the agents' antigens are processed, though, by the macrophages and presented preferentially in association with class II MHC molecules to CD4 + class II MHC-restricted TH cells, which become stimulated to secrete interferon- ⁇ , which, in turn, activates macrophages. Such activation results in the cells' exhibiting increased bacteriocidal ability.
  • TH cells are composed of at least two distinct subpopulations, termed TH1 and TH2 cell subpopulations.
  • Evidence suggests that TH1 and TH2 subtypes represent extremely polarized populations of TH cells. While such subpopulations were originally discovered in murine systems (reviewed in Mosmann, T.R. and Coffman, R.L., 1989, Ann. Rev. Immunol. 7:145), the existence of TH1- and TH2-like subpopulations has also been established in humans (Del Prete, A.F. et al., 1991, J. Clin. Invest. 88:346; Wiernenga, E.A. et al., 1990, J. Imm. 144:4651; Yamamura, M.
  • THl-like and TH2-like cells can represent the most extremely polarized TH cell subpopulations
  • other TH cell subpopulations such as TH0 cells (Firestein, G.S. et al., 1989, J. Imm. 143 :518) , which represent TH cells which have characteristics of TH1 and TH2 cell subpopulations.
  • THl-like and TH2-like cells appear to function as part of the different effector functions of the immune system (Mosmann, T.R. and Coffmann, R.L., 1989, Ann. Rev. Imm. 7:145). Specifically, THl-like cells direct the development of cell-mediated immunity, triggering phagocyte-mediated host defenses, and are associated with delayed hypersensitivity . Accordingly, infections with intracellular microbes tend to induce THl-type responses. TH2 cells drive humoral immune responses, which are associated with, for example, defenses against certain helminthic parasites, and are involved in antibody and allergic responses.
  • TH1 cells are known to secrete interleukin-2 (IL-2) , interferon- ⁇ (IFN- ⁇ ) , and lymphotoxin, while TH2 cells secrete interleukin-4 (IL-4) , interleukin-5 (IL-5) , and interleukin-10 (IL-10) .
  • IL-2 interleukin-2
  • IFN- ⁇ interferon- ⁇
  • IL-4 interleukin-4
  • IL-5 interleukin-5
  • IL-10 interleukin-10
  • TH1 and TH2 subpopulations arise from a common naive precursor (referred to as THP) .
  • THP naive CD4 + cells from mice which express a single transgenic T cell receptor can be induced to develop into either the TH1 or TH2 cell type.
  • the conditions of antigen stimulation including the nature and amount of antigen involved, the type of antigen-presenting cells, and the type of hormone and cytokine molecules present seem to all represent determinants of the pattern of TH1 versus TH2 differentiation, with, perhaps, the decisive role belonging to the cytokines present. With such a complex series of possible determinants, a full accounting of the exact factors important in driving TH1 or TH2 differentiation are, as yet largely unknown.
  • CD8 + CTLs can, under certain conditions, also exhibit THl-like or TH2-like cytokine profiles (Seder, R.A. et al., 1995, J. Exp. Med. 181:5-7; Manetti, R. et al., 1994, J. Exp. Med. 180:2407-2411; Maggi, E. et al., 1994, J. Exp. Med. 180:489-495) . While the precise functional role of such CD8 + TH-like cells is currently unknown, these cell subpopulations appear to have great relevance to immune responses against infectious agents such as viruses and intracellular parasites.
  • TH1 and TH2 subpopulations tend to negatively regulate one another through the actions of cytokines unique to each.
  • TH1- produced IFN- ⁇ negatively regulates TH2 cells
  • TH2- produced IL-10 negatively regulates TH1 cells.
  • cytokines produced by TH1 and TH2 antagonize the effector functions of one another (Mosmann, T.R. and Moore, 1991, Immunol. Today 12:49).
  • Such disorders can include, for example, atopic conditions (i.e. , IgE-mediated allergic conditions) such as asthma, allergy, including allergic rhinitis, dermatitis, including psoriasis, pathogen susceptibilities, chronic inflammatory disease, organ- specific autoimmunity, graft rejection and graft versus host disease.
  • atopic conditions i.e. , IgE-mediated allergic conditions
  • asthma i.e. , IgE-mediated allergic conditions
  • dermatitis including psoriasis, pathogen susceptibilities
  • chronic inflammatory disease organ-specific autoimmunity
  • graft rejection graft versus host disease.
  • nonhealing forms of human and murine leishmaniasis result from strong but counterproductive TH2- like-dominated immune responses.
  • Lepromatous leprosy also appears to feature a prevalent, but inappropriate, TH2-like response.
  • TH2-like clones appear to be more efficient supporters of HIV viral replication than THl-like clones.
  • THl-mediated inflammatory responses to many pathogenic microorganisms are beneficial, such responses to self antigens are usually deleterious. It has been suggested that the preferential activation of THl-like responses is central to the pathogenesis of such human inflammatory autoimmune diseases as multiple sclerosis and insulin-dependent diabetes.
  • THl-type cytokines predominate in the cerebrospinal fluid of patients with multiple sclerosis, pancreases of insulin-dependent diabetes patients, thyroid glands of Hashimoto's thyroiditis, and gut of Crohn ' s disease patients, suggesting that such patients mount a THl-like, not a TH2-like, response to the antigen (s) involved in the etiopathogenesis of such disorders.
  • the ability to identify those genes which are differentially expressed within and/or among such TH cell subpopulations is required to achieve such a goal.
  • investigations have focused on the expression of a limited number of specific known cytokines and cytokine receptors in the TH cell population. Cytokines, however, exert effects on cell types in addition to specific TH cell subpopulations, i.e. , exhibit a variety of pleiotropic effects. It would be beneficial, therefore, to identify reliable markers (e.g. , gene sequences) of TH cell subpopulations whose effects are TH cell subpopulation specific, e.g. , which, unlike secreted cytokines, are TH cell subpopulation specific.
  • the present invention relates to methods and compositions for the treatment of immune disorders, especially T helper (TH) cell and TH cell-like related disorders.
  • the present invention additionally relates to methods and compositions for treating, ameliorating or modulating ischemic disorders or injuries or mast cell- related processes or disorders.
  • genes are identified and described which are differentially expressed within and among TH cells and TH cell subpopulations.
  • genes are identified and described which are differentially expressed within TH cell subpopulations in TH cell subpopulation-related disorders.
  • the modulation of the expression of the identified genes and/ or the activity of the identified gene products can be utilized therapeutically to ameliorate immune disorder symptoms and to modulate TH cell responsiveness, for example, responsiveness to antigen.
  • the identified genes and/or gene products can be used to diagnose individuals exhibiting or predisposed to such immune disorders.
  • the identified genes and/or gene products can be used to detect TH cell responsiveness, for example, responsiveness to antigen.
  • Differential expression refers to both quantitative as well as qualitative differences in the genes' temporal and/or cellular expression patterns within and among the TH cell subpopulations. Differentially expressed genes can represent "fingerprint genes” and/or "target genes”.
  • Fingerprint gene refers to a differentially expressed gene whose expression pattern can be utilized as part of a prognostic or diagnostic evaluation of immune disorders, e.g. , TH cell-related disorders, or which, alternatively, can be used in methods for identifying compounds useful in the treatment of such disorders.
  • immune disorders e.g. , TH cell-related disorders
  • the effect of the compound on the fingerprint gene expression normally displayed in connection with the disorder can be used to evaluate the efficacy of the compound as a treatment for such a disorder, or may, additionally, be used to monitor patients undergoing clinical evaluation for the treatment of such disorders.
  • Fingerprint pattern refers to the pattern generated when the expression pattern of a series (which can range from two up to all the fingerprint genes which exist for a given state) of fingerprint genes is determined. A fingerprint pattern can be used in the same diagnostic, prognostic, and compound identification methods as the expression of a single fingerprint gene.
  • Target gene refers to a differentially expressed gene involved in immune disorders, e.g. , TH cell related disorders, such that modulation of the level of target gene expression or of a target gene product activity can act to ameliorate the immune disorder.
  • Target gene expression or activity of the target gene product can be used in the treatment of immune disorders.
  • pathway genes are defined via the ability of their gene products to interact with gene products involved in TH cell subpopulation-related disorders and/or to interact with gene products which are involved in the differentiation and effector function of the TH cell subpopulations. Pathway genes can also exhibit target gene and/or fingerprint gene characteristics.
  • target, fingerprint and/or pathway genes described herein can be differentially expressed within and/or among TH cell subpopulations, and/or can interact with TH cell subpopulation gene products, the genes can also be involved in mechanisms important to additional immune processes.
  • the invention encompasses the following nucleotides, host cells expressing such nucleotides and the expression products of such nucleotides: (a) nucleotides that encode a mammalian differentially expressed and/or pathway gene product including, but not limited to a human and murine 10, 54, 57, 105, 106, 161 and 200 gene product; (b) nucleotides that encode portions of a differentially expressed and/or pathway gene product that corresponds to its functional domains, and the polypeptide products encoded by such nucleotide sequences, and in which, in the case of receptor- type gene products, such domains include, but are not limited to extracellular domains (ECD) , transmembrane domains (TM) and cytoplasmic domains (CD) ; (c) nucleotides that encode mutants of a differentially expressed and/or pathway gene product, in which all or part of one of its domains is deleted or altered, and which, in the case of receptor-type gene products, such mutants
  • the present invention also includes the products of such fingerprint, target, and pathway genes, as well as antibodies to such gene products. Furthermore, the engineering and use of cell- and animal-based models of TH cell subpopulation- related disorders to which such gene products can contribute, are also described.
  • the present invention also relates to methods for prognostic and diagnostic evaluation of various TH cell subpopulation-related disorders, and for the identification of subjects who are predisposed to such disorders. Furthermore, the invention provides methods for evaluating the efficacy of drugs for immune disorders, and monitoring the progress of patients involved in clinical trials for the treatment of such disorders.
  • the TH cell subpopulation-related disorders described herein can include, for example, TH1 or THl-like related disorders or can, alternatively, include TH2 or TH2-like related disorders.
  • TH1 or THl-like related disorders include chronic inflammatory diseases and disorders, such as Crohn 's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ- specific autoimmunity, including multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease and sarcoidosis.
  • TH2 or TH2-like related disorders include atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glo erular nephritis, certain pathogen susceptibilities such as helminthic (e.g. , leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.
  • helminthic e.g. , leishmaniasis
  • certain viral infections including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.
  • Ischemic disorder or injury can be treated via the methods of the invention.
  • ⁇ Ischemic disorder or injury refers to any disorder or injury to tissues or organs which results from local deficiency of the blood supply and/or of said tissue or organs. Such deficiencies of the blood supply and/or hypoxia are generally produced by a restriction or obstruction of the blood supply to said tissue or organs.
  • Ischemic disorders or injuries which may be treated by the methods of the present invention include, but are by no means limited to, ischemic renal disease or injury, or myocardial ischemia such as angina pectoris.
  • Ischemic disorders or injuries which may be treated by the methods of the present invention also include damage or injury to tissue or organs due to an infarction, such as damage to the heart, brain (such as in a stroke) , spleen, kidney, intestine, lung, and testes.
  • the methods of the invention may also be used to regulate the extent or degree of ischemic injury in other tissues, such as tumor tissues including, but not limited to, tumors of the uterus and ovaries.
  • the ischemic disorders or injuries which may be treated by the methods of the present invention still further include ischemic injury or damage to transplanted organs which occurs during transplant.
  • the methods and compositions described herein can be utilized in the prognostic and diagnostic evaluation of disorders involving other immune cells, including CD8 + CTLs, exhibiting TH-like cell subpopulation gene expression patterns and/or activity. It is still further contemplated that the methods and compositions described herein can be utilized in the amelioration of symptoms stemming from disorders involving such immune cells, especially such CD8 + CTLs, which exhibit TH-like cell subpopulation gene expression patterns and/or activity.
  • the invention further provides methods for the identification of compounds which modulate the expression of genes or the activity, e.g. level, of gene products involved in TH cell subpopulation-related disorders and processes relevant to the differentiation, maintenance and/or effector function of the subpopulations.
  • methods for identifying compounds which affect the level of 103 gene expression and/or gene product are presented herein.
  • the present invention provides methods for identifying compounds which bind to gene products of the differentially expressed sequences identified herein.
  • methods include, but are not limited to, methods for identifying compounds which bind to a 103 gene product.
  • the present invention provides methods for the treatment of TH cell subpopulation-related disorders which can, for example, involve the administration of such modulatory compounds to individuals exhibiting TH cell subpopulation-related disorder symptoms or tendencies. Additionally, treatment can result in the stimulation or depletion of one or more of the TH cell subpopulations.
  • stimulation can refer to an effective increase in the number of cells belonging to a TH cell subpopulation, via, for example, the proliferation of such TH cell subpopulation cells. The term can also refer to an increase in the activity of cells belonging to a TH cell subpopulation, as would be evidenced, for example, by a per cell increase in the expression of the TH cell subpopulation- specific cytokine pattern.
  • Depletion can refer to an effective reduction in the number of cells belonging to a TH cell subpopulation, via, for example, a reduction in the proliferation of such TH cell subpopulation cells.
  • the term can also refer to a decrease in the activity of cells belonging to a TH cell subpopulation, as would be evidenced, for example, by a per cell decrease in the expression of the TH cell subpopulation-specific cytokine pattern.
  • the methods a compositions described herein can also be used in the treatment of ischemic disorders or injuries.
  • presented herein are methods of using the 200 gene, its gene product, and antibodies thereto to treat or regulate ischemic disorders and/or injuries.
  • the genes or gene products of the invention may be administered to an individual so as to ameliorate the symptoms of the ischemic disorder or injury.
  • compounds, such as specific antibodies, including monoclonal antibodies, which bind specifically to the genes or gene products of the present invention and modulate their expression or activity may also be administered to an individual suffering from an ischemic disorder or injury.
  • the invention is based, in part on systematic search strategies involving paradigms which utilize TH0, TH1, TH2 , THl-like and TH2-like cells, in systems which mimic the activity of the immune system or immune disorders, coupled with sensitive and high-throughput gene expression assays, to identify genes differentially expressed within and/or among TH cell subpopulations.
  • the search strategies and assays used herein permit the identification of all genes, whether known or novel, which are dif erentially expressed within and among TH cell subpopulations, as well as making possible the characterization of their temporal regulation and function in the TH cell response and/or in TH cell mediated disorders.
  • This comprehensive approach and evaluation permits the discovery of novel genes and gene products, as well as the identification of a constellation of genes and gene products (whether novel or known) involved in novel pathways (e.g. , modulation pathways) that play a major role in the TH-cell mediated immune responses and TH cell subpopulation-related disorders.
  • the present invention makes possible the identification and characterization of targets useful for prognosis, diagnosis, monitoring, rational drug design, and/or therapeutic intervention of immune system disorders.
  • Section 9 describes the successful cloning of a human homolog of one of the identified genes (the 200 gene) .
  • the 102 and 103 genes represent genes which, while previously known, are shown here to be differentially expressed among TH cell subpopulations.
  • the 102 gene corresponds to the Granzyme A, or Hanukah factor, gene, which encodes a trypsin-like serine protease. While this gene had previously been reported to be expressed in natural killer cells and a fraction of CD4 + cells, the results described herein reveal, for the first time, that the gene is differentially expressed within the TH2 cell subpopulation. Specifically, the 102 gene is expressed at a level many-fold higher in the TH2 cell subpopulation than in the TH1 cell subpopulation.
  • the 103 gene corresponds to a gene known as the Tl, ST-2 or Fit-1 gene, which encodes, possibly via alternative splicing, both transmembrane and soluble gene products.
  • the gene 103 products belong to the immunoglobulin superfamily, and bear a high resemblance to the interleukin-1 (IL-1) receptor.
  • IL-1 interleukin-1
  • the results presented herein demonstrate, for the first time, that this gene is expressed, in vivo, in a tightly controlled TH2-specific fashion.
  • the gene 103 products can be utilized in a variety of methods to diagnose and/or modulate immune system disorders, in particular TH2 cell subpopulation- related disorders.
  • results, including results obtained in vivo in an animal model for asthma, a TH2-like disorder are presented herein which indicate that the 103 gene product provides a critical signal to TH2 effector cells.
  • the 54, 105, 106 and murine 200 genes are each shown to be differentially expressed within the TH1 cell subpopulation. Specifically, these genes are expressed at levels many-fold higher in TH1 cell subpopulations than in TH2 cell subpopulations.
  • the novel 54 gene product is a 371 amino acid cysteine protease, as evidenced by the presence of three thiol protease domains at approximately amino acid residue 145 to 156 (CYS domain) , approximately amino acid residue 287 to 297 (HIS domain) and approximately amino acid residue 321 to 340 (ASN domain) of the 54 gene product amino acid sequence.
  • the 10 and 57 genes represent TH inducible gene sequences. That is, the expression of such genes in unstimulated TH cells is either undetectable or barely detectable, but is significantly upregulated in both stimulated TH1 and stimulated TH2 cells.
  • the 10 and 57 genes and/or their gene products can represent new targets for therapeutic treatment as part of a non-TH cell subpopulation dependent intervention program.
  • the 10 gene product is a 338 amino acid receptor molecule which is a particularly suitable target for such a program in that the 10 gene product belongs to a class of proteins having a seven transmembrane domain sequence motif, which tend to represent G protein-coupled receptor molecules.
  • the 10 gene product structure therefore, indicates that it may be involved in signal transduction events which may be important to T cell responses in general, and further indicates that modulation of 10 gene product may effectively ameliorate a wide range of T cell-related disorders.
  • the 10 gene product is a transmembrane product, its activity, via either a physical change in the number of 10 gene-expressing cells or by a change in the functional level of 10 gene product activity, can be particularly amenable to modulation.
  • natural ligands, derivatives of natural ligands and antibodies which bind to the 10 gene product can be utilized to reduce the number of induced T cells present by either physically separating such cells away from other cells in a population, or, alternatively, by targeting the specific destruction of the induced T cells or inhibiting the proliferation of such T cells.
  • compounds such as 10 gene sequences or gene products can be utilized to reduce the level of induced T cell activity, and, ultimately, bring about the amelioration of a wide range of T cell-related disorders.
  • the compounds can compete with the endogenous (i.e. , natural) ligand for the 10 gene product, leading to a modulation of induced T cell activity.
  • Soluble proteins or peptides, such as peptides comprising one or more of the extracellular domains, or portions and/or analogs thereof, of the 10 gene product including, for example, soluble fusion proteins such as Ig- tailed fusion proteins, can be particularly useful for this purpose.
  • antibodies directed against one or more of the extracellular portions of the 10 gene product may either reduce 10 gene product function by, for example, blocking ligand binding.
  • antibodies directed against the 10 gene product can, in certain instances, serve to increase the level of 10 gene product activity.
  • the receptor nature of the 10 gene product makes possible useful methods for the identification of compounds which modulate the receptor's functional activity and which can act as therapeutic agents in the amelioration of a wide range of T cell-related disorders.
  • functional assays which measure intracellular calcium release levels may be utilized to identify compounds which act as either agonists or antagonists of 10 gene product activity.
  • Such assays may, additionally, be utilized to identify the natural 10 gene product ligand.
  • any of these modulatory compounds can be utilized as therapeutic agents for the amelioration of a wide range of T cell-related disorders.
  • the 161 gene is shown to be an additional new and potentially interesting target for a therapeutic method aimed at the amelioration of immune disorder related symptoms.
  • 161 gene expression may be indicative of the presence of yet another TH cell subpopulation, in addition to TH1, TH2 and TH0 cell subpopulations .
  • the identification of TH cell subpopulation specific markers can be utilized in the treatment of a number of immune disorders, especially TH cell subpopulation-related disorders.
  • markers for the TH2 subpopulation can be used to ameliorate conditions involving an inappropriate IgE immune response, including but not limited to the symptoms which accompany atopic conditions such as allergy and/or asthma.
  • IgE-type antibodies are produced by stimulated B cells which require, at least in part, IL-4 produced by the TH2 cell subpopulation. Therefore, a treatment which reduces the effective concentration of secreted IL-4, e.g. , by reducing the activity or number of TH2 cells, will bring about a reduction in the level of circulating IgE, leading, in turn, to the amelioration or elimination of atopic conditions.
  • Any of the TH2-specific gene products described herein can, therefore, be used as a target to reduce or deplete the number and/or activity of TH2 cell subpopulation cells for the treatment of such conditions.
  • the 103 gene can be particularly suitable for this purpose since one of its gene products is a membrane-bound TH2 cell subpopulation molecule. Accordingly, natural ligands, derivatives of natural ligands and antibodies which bind to this 103 gene product, can be utilized to reduce the number of TH2 cells present by either physically separating such cells away from other cells in a population, or, alternatively, by targeting the specific destruction of TH2 cells or inhibiting the proliferation of such TH2 cells. Additionally, compounds such as 103 gene sequences or gene products can be utilized to reduce the level of TH2 cell activity, cause a reduction in IL-4 production, and, ultimately, bring about the amelioration of IgE and/or TH2- related disorders.
  • the compounds can compete with the endogenous (i.e. , natural) ligand for the 103 gene product.
  • the resulting reduction in the amount of ligand- bound 103 gene transmembrane protein will modulate TH2 cellular activity.
  • Soluble proteins or peptides, such as peptides comprising the extracellular domain, the secreted form, or portions and/or analogs thereof, of the 103 gene product including, for example, soluble fusion proteins such as Ig-tailed fusion proteins, can be particularly useful for this purpose.
  • antibodies directed against the 103 gene product such as directed against the extracellular domain of the 103 gene product, can be utilized for this purpose.
  • TH cell subpopulation specific markers can additionally be utilized in the treatment of a TH1 cell subpopulation-related disorders.
  • markers for the TH1 cell subpopulation can be used to ameliorate conditions involving an inappropriate cell- mediated immune response, including, but not limited to chronic inflammatory and autoimmune disorders.
  • transgenic animals overexpressing or misexpressing such gene sequences and/or transgenic "knockout" animals exhibiting little or no expression of such sequences can be utilized as animal models for TH cell subpopulation-related disorders.
  • TH1 cell subpopulation specific gene sequences and/or gene products such as the 54 (which encodes a 371 amino acid cysteine protease gene product) , 105, 106 and 200 (the murine homolog of which encodes a 280 amino acid transmembrane gene product, the human homolog of which encodes a 301 amino acid transmembrane gene product, both of which are members of the Ig superfamily) genes can, therefore, be suitable for ameliorating such THl cell subpopulation-related disorders.
  • the 200 gene product can be particularly suitable for such a purpose in that it is not only THl cell subpopulation- restricted, but the Ig superfamily 200 gene product is, additionally, membrane-bound.
  • natural ligands, derivatives of natural ligands and antibodies which bind to the 200 gene product can be utilized to reduce the number of THl cells present by either physically separating such cells away from other cells in a population, or, alternatively, by targeting the specific destruction of THl cells or inhibiting the proliferation of such THl cells.
  • compounds such as 200 gene sequences or gene products such as soluble 200 gene products, can be utilized to reduce the level of TH2 cell activity, thus bringing about the amelioration of THl cell subpopulation-related disorders.
  • the compounds can compete with the endogenous (i.e.. natural) ligand for the 200 gene product.
  • Soluble proteins or peptides such as peptides comprising the extracellular domain, or portions (such as, for example, the Ig portion) and/or analogs thereof, of the 200 gene product, including, for example, soluble fusion proteins such as Ig-tailed fusion proteins, can be particularly useful for this purpose.
  • soluble fusion proteins such as Ig-tailed fusion proteins
  • Example presented in Section 12, below describes successful use of antibodies directed against the 103 gene product as well as 103 /Ig fusion proteins to ameliorate symptoms of asthma in an accepted animal model for the TH2-related disorder.
  • the results indicate that the 103 gene product provides a critical signal to TH2 cells and can successfully be used as a target for selective modulation of TH immune responses (e.g. , for selective suppression of TH2 immune responses and/or selective enhancement of THl immune responses) .
  • the invention is also based, in part, on the discovery that among the genes and gene products described herein are ones also involved in processes related to tissue repair and remodeling after injury, particularly after ischemic injury.
  • the example presented in Section 13, below demonstrates the successful use of antibodies which bind to the extracellular domain of the 200 gene product to inhibit repair of ischemic kidney injury.
  • the invention also makes possible the treatment of ischemic disorders and injuries.
  • the invention is also based, in part, on the discovery that the 103 gene is expressed in mast cells, as demonstrated in the Example presented in Section 14, below.
  • TH cell subpopulation refers to a population of TH cells exhibiting a gene expression pattern (e.g.. a discrete pattern of cytokines and/or receptor or other cell surface molecules) and activity which are distinct from the expression pattern and activity of other TH cells.
  • a gene expression pattern e.g.. a discrete pattern of cytokines and/or receptor or other cell surface molecules
  • Such TH cell subpopulations can include, but are not limited to, TH0, THl and TH2 subpopulations, which will, for clarity and example, and not by way of limitation, be frequently used herein as representative TH cell subpopulations .
  • TH-like cell subpopulation e.g.
  • THl-like or “TH2-like”) as used herein is intended to refer not only to a population of CD4 + TH cells having the properties described, above, for a TH cell subpopulation, but also refers to CD4 ⁇ cells, including CD8 + CTLs, which exhibit TH- like cytokine expression patterns.
  • Target gene refers to a differentially expressed gene involved in immune disorders and/or in the differentiation, maintenance and/or effector function of TH cell subpopulations, such that modulation of the level of target gene expression or of target gene product presence and/or activity can, for example, act to result in the specific depletion or repression, or, alternatively, the stimulation or augmentation of one or more TH cell subpopulation, bringing about, in turn, the amelioration of symptoms of immune disorders, e.g.. TH cell subpopulation- related disorders.
  • a target gene can also exhibit fingerprint and/or pathway gene characteristics.
  • Fingerprint gene refers to a differentially expressed gene whose mRNA expression pattern, protein level and/or activity can be utilized as part of a prognostic or diagnostic in the evaluation of immune disorders, e.g. , TH cell subpopulation-related disorders, or which, alternatively, can be used in methods for identifying compounds useful for the treatment of such disorders, by, for example, evaluating the effect of the compound on the fingerprint gene expression normally displayed in connection with the disease.
  • a fingerprint gene can also exhibit target and/or pathway gene characteristics.
  • Fingerprint pattern refers to the pattern generated when tne mRNA expression pattern, protein level and/or activity of a series (which can range from two up to all the fingerprint genes which exist for a given state) of fingerprint genes is determined.
  • a fingerprint pattern can be a part of the same methods described, above, for the expression of a single fingerprint gene.
  • Pathway genes refers to a gene whose product exhibits an ability to interact with gene products involved in immune disorders, e.g , TH cell subpopulation- related disorders and/or to interact with gene products which are involved in the differentiation and effector function of TH cell subpopulations. Pathway genes can also exhibit target gene and/or fingerprint gene characteristics.
  • “Negative modulation”, as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
  • the term, as used herein refers to a reduction in the number and/or activity of cells belonging to the TH cell subpopulation relative to the number and/or activity of the TH cell subpopulation in the absence of the modulatory treatment.
  • “Positive modulation”, as used herein, refers to an increase in the level and/or activity of target gene product relative to the level and/or activity of the gene product in the absence of the modulatory treatment.
  • the term, as used herein refers to an increase in the number and/or activity of cells belonging to the TH cell subpopulation, relative to the number and/or activity of the TH cell subpopulation in the absence of the modulatory treatment.
  • Ischemic disorder or injury refers to any disorder or injury to tissues or organs which results from local deficiency of the blood supply and/or hypoxia to said tissue or organs. Such deficiency of the blood supply and/or hypoxia is generally produced by a restriction or obstruction of the blood supply to said tissue or organs.
  • mast-cell related processes or disorders includes, but is not limited to, atherosclerosis and myocardial ischemia/reperfusion.
  • FIG. 1 Differential display analysis of RNA from murine TH cell subsets. Splenic T cells derived from T cell receptor transgenic mice were differentiated in vitro to become polarized populations of THl or TH2 subtypes. Lane 1: TH2 population 24 hours after tertiary stimulation; lane 2: THl population 24 hours after tertiary stimulation; lane 3: TH2 population 1 week after secondary stimulation; lane 4: THl population 1 week after secondary stimulation; lane 5: TA3 cell line, which was used as antigen presenting cell (APC) for in vitro stimulation.
  • APC antigen presenting cell
  • Each set of lanes consists of duplicates (a and b) , in which cDNAs were independently generated from the same source of RNA. Arrow points to differentially expressed sequence, which is referred to herein as band 102. Further, the gene corresponding to band 102 is referred to herein as the 102 gene. All lanes are products of a polymerase chain reaction (PCR) in which T X1 GG was used as the 3 ' oligonucleotide and a random lO er oligonucleotide (Oligo #4, OP-D kit, Operon, Inc.) was used as the 5' oligonucleotide.
  • PCR polymerase chain reaction
  • FIG. 2 Nucleotide sequence of clone 102.1 of band 102 (SEQ. ID NO: 1) .
  • the gene corresponding to band 102 is referred to herein as the 102 gene.
  • FIG. 3 Northern blot analysis of confirming differential regulation of the 102 gene within primary TH1/TH2 cultures and murine tissues.
  • RNA was harvested from T cell lines derived from a T cell receptor transgenic strain stimulated in vitro. Lane 1, TH2 , 40 hours after second stimulation; lane 2, THl, 40 hours after second stimulation; lane 3, TH2 population 24 hours after tertiary stimulation; lane 4, THl, 24 hours after tertiary stimulation; lane 5, murine thymus; lane 6, murine spleen. Five microgra s of total RNA was used per lane. The cloned band 102 sequence was used as a probe.
  • FIG. 4A Nucleotide sequence clone 103.1 of band 103 (SEQ ID NO: 2) .
  • the gene corresponding to band 103 is referred to herein as gene 103.
  • FIG. 4B 103 gene products.
  • This diagram illustrates the relationship between the sequence encoded by band 103, 103 gene (also known as ST-2 , Tl and Fit-1) products and the IL-1 receptor polypeptide structure. The extracellular, transmembrane and cytoplasmic domains of the proteins are noted, along with the amino acid residues marking the boundaries of these domains. (Adapted from Yanagisawa et al., 1993, FEBS Lett. 318:83-87.)
  • FIG. 4C The nucleotide sequence encoding the secreted form of murine 103 gene product is depicted (SEQ ID NO: 49; Accession No. E07714) .
  • FIG. 4D The amino acid sequence of the secreted form of murine 103 is depicted (SEQ ID NO: 47; Accession No. P14719) .
  • FIG. 4E The nucleotide sequence encoding the transmembrane murine 103 gene product is depicted (SEQ ID NO:38; Accession No. E08652).
  • FIG. 4F The amino acid sequence of the transmembrane receptor of murine 103 is depicted (SEQ ID NO: 39; Accession No. S29498) .
  • the extracellular domain of the full length, transmembrane product extends from amino acid residue 1 to 342 of SEQ ID NO: 38 (SEQ ID NO:41), the transmembrane domain of the full length, transmembrane product extends from amino acids 343 to 366 of SEQ ID NO: 38 (SEQ ID NO: 48) the intracellular domain of the full length, transmembrane product extends from amino acid residues 367 to 567 of SEQ ID NO: 38 (SEQ ID NO:43) .
  • FIG. 4G The nucleotide sequence encoding the secreted product of the human 103 gene is depicted (SEQ ID NO: 44; Accession No. NM 003856) .
  • FIG. 4H The amino acid sequence of the secreted product of the human 103 gene is depicted (SEQ ID NO: 45; Accession No. NM_003856) .
  • FIG. 5 Quantitative RT-PCR analysis of 103 gene expression in polarized populations of murine TH cells. RNA samples were harvested from cultured T cell populations 24 hours after tertiary stimulation with antigen. cDNA samples were PCR amplified and the products of those reactions were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining. 103 gene expression is shown in the upper panel. ⁇ -actin data, bottom panel, was included as a control for differences in sample quality. The numbers above each lane represent the dilution factors of each cDNA. The same cDNA samples were used for both the 103 gene and the ⁇ -actin amplifications.
  • FIG. 6 Northern blot analysis of 103 gene expression in representative murine TH cell lines (TH2: CDC25, D10.G4, DAX; THl: AE7.A, Dorris, Dl.l). Clones were either unstimulated (-) or stimulated (+) for 6 hours with plate- bound anti-CD3 antibody. Ten micrograms of total RNA were loaded per lane. The positions of 18s and 28s ribosomal RNA are shown as reference markers.
  • FIG. 7 Northern blot analysis of 103 gene expression in T cell clones and murine tissues.
  • Lane 1 DAX cells, no stimulation; lane 2, AE7 cells, stimulation; lane 3, AE7 cells, no stimulation; lane 4, D10.G4 cells, stimulation; lane 5, D10.G4 cells, no stimulation; lane 6, brain; lane 7, heart; lane 8, lung; lane 9, spleen; lane 10, liver.
  • Clones were stimulated with plate-bound anti-CD3 antibody for 6 hours. 7.5 and 10 micrograms total RNA was used for each cell line and each tissue, respectively.
  • a, b, and c arrows refer to RNA encoding full length (a) and truncated (b,c) forms of the 103 gene.
  • FIG. 8 RNAse protection analysis of 103 gene mRNA, illustrating regulation of 103 gene expression in murine TH cell clones.
  • Lanes 2-6 ⁇ -actin protection; lanes 9-13: 103 gene protection; lanes 1 and 8: markers; lanes 2 and 9: unstimulated THl clones; lanes 3 and 10: stimulated THl clones; lanes 4 and 11: unstimulated TH2 clones; lanes 5 and 12: stimulated TH2 clones; lanes 6 and 13: fully RNAse A digested unprotected probe; lanes 7 and 14: probe alone, in absence of added RNAse.
  • FIG. 9 The full length 10 gene nucleotide sequence (SEQ ID NO: 3) is shown on the top line, while the derived amino acid sequence of the 10 gene product (SEQ ID NO: 9) is shown on the bottom line.
  • the underlined portion of the nucleotide sequence corresponds to the band 10 nucleotide sequence.
  • the data shown in FIG. 10A-C was obtained through the use of the portion of the 10 gene product which is encoded by the band 10 nucleotide sequence.
  • FIG. 10A-C 10 gene hydrophilicity data, indicating that the 10 gene-derived amino acid sequence predicts the presence of a seven transmembrane domain structural motif.
  • 10A platelet activating factor receptor hydrophilicity plot illustrating the protein's seven transmembrane domain structural motif
  • 10B 10 gene hydrophilicity plot illustrating a portion of the protein's putative seven transmembrane domain structural motif
  • 10C platelet activating factor receptor hydrophilicity plot illustrating part of the protein's seven transmembrane structural motif.
  • FIG. 11 Chromosomal mapping of locus containing the 10 gene sequence. A map of a portion of mouse chromosome 12 is shown. Numbers to left of chromosome are in centiMorgans; D12NDS11, D12MIT4, and D12MIT8 represent mouse microsatellite markers; TH10 represents 10 gene.
  • FIG. 12 Nucleotide sequence of clone 7 of band 57 (SEQ ID NO: 4). The gene corresponding to band 57 is referred to herein as the 57 gene.
  • FIG. 13 Consensus nucleotide sequence of band 105 (SEQ ID NO: 5). "N” signifies "any nucleotide”. The gene corresponding to band 105 is referred to herein as the 105 gene.
  • FIG. 14 Nucleotide sequence obtained from clone H of band 106 (SEQ ID NO:6). "N” signifies "any nucleotide”. The gene corresponding to band 106 is referred to herein as the 106 gene.
  • FIG. 15 Nucleotide sequence of clone G of band 161 (SEQ ID NO:7).
  • the gene corresponding to band 161 is referred to herein as the 161 gene.
  • FIG. 16 Multiple sequence alignment of 161 clone G with amino acid sequences identified in a BLAST search. Asterisks signify positions that are identical; dots indicate conserved positions.
  • FIG. 17 Nucleotide and amino acid sequence of the full length murine 200 gene.
  • Bottom line murine 200 gene nucleotide sequence (SEQ ID N0:8); top line: murine 200 gene product derived amino acid sequence (SEQ ID NO: 10) .
  • FIG. 18 Northern blot analysis of murine 200 gene expression in representative murine TH cell lines (TH2: CDC25, D10.G4, DAX; THl: AE7.A, Dorris, Dl.l). Clones were either unstimulated (-) or stimulated (+) for 6 hours with plate-bound anti-CD3 antibody. The positions of RNA markers, in kilobases, are shown for reference. The arrow marks the position of 200 gene mRNA.
  • FIG. 19 Northern blot analysis of 54 gene expression within THl (Dl.l, Dorris, AE7) cell lines and TH2 (D10.G4, DAX, CDC25) cell lines, either stimulated (+) or unstimulated (-) with anti-CD3 antibodies. 15 micrograms of total RNA were loaded per lane. Cells were stimulated between 6 and 7 hours with anti-CD3 antibodies, as described, below, in Section 8.1. The Northern blots were hybridized with a probe made from the entire band 54 nucleotide sequence.
  • FIG. 20 Northern blot analysis of gene 54 time course study. RNA from THl cell line AE7 cells was isolated, either unstimulated or stimulated for varying periods of time, as indicated. Second, RNA from two TH2 cell lines (DAX, CDC25) was isolated from either unstimulated cells or from cells which had been stimulated for two hours with anti-CD3 antibodies. 15 micrograms total RNA were loaded per lane. A band 54 DNA probe was used for hybridization.
  • FIG. 21 Northern blot analysis of 54 gene expression in various tissues. 15 micrograms of total RNA were loaded per lane. A band 54 DNA probe was used for hybridization.
  • FIG. 22 Nucleotide and amino acid sequence of the full length 54 gene.
  • Bottom line 54 gene nucleotide sequence (SEQ ID NO: 11).
  • Top line 54 gene derived amino acid sequence (SEQ ID NO: 12).
  • FIG. 23 The 54 gene product bears a high level of homology to the cysteine protease class of proteins.
  • the 54 gene product amino acid is depicted with its predicted pre- pro sequence and mature cysteine protease polypeptide sequence identified.
  • the individual boxed amino acid residues represent residues thought to lie within the cysteine protease active site and the stretch of amino acid residues which are boxed represent a region with homology to a stretch of amino acid residues normally seen within the preproenzyme portion of cysteine protease molecules.
  • the circled amino acid residues within this stretch represent conserved amino acids.
  • the arrow indicates the putative post-translational cleavage site.
  • FIG. 24 Nucleotide and amino acid sequence of the full length human 200 gene. Bottom line: human 200 gene nucleotide sequence (SEQ ID NO: 23) ; top line: human 200 gene product derived amino acid sequence (SEQ ID NO:24).
  • FIG. 25 Flow cytometry data demonstrates that the 3E10 mAb recognizes and binds to representative clones of the TH2 cell subpopulation (D10.G4; DAX), but not clones of the THl subtype (AE7 ; Dorris) .
  • the graphs in this figure present the results of the flow cytometry analyses by depicting the number of cells exhibiting a given level of fluorescence. Staining above background levels represents antigen-specific binding and, therefore, the presence of cell surface 103 gene product. The further to the right the peaks are shifted, the greater the staining intensity, and therefore antibody binding, exhibited by a cell population.
  • Fig. 26 Analysis of the cytokine profile in mouse BAL.
  • the data presented in this figure reveals high levels of IL- 4, IL-5, IL-6, IL-10 and IL-13 in TH2 recipient OVA challenged mice (closed bars) .
  • Pretreatment with 3E10 mAb resulted in a dramatic reduction in IL-4, IL-5, IL-6 and IL-13, but had no effect on IL-10 levels in the BAL (open bars) .
  • OVA challenge of THl recipient mice resulted in high levels of IFN- ⁇ in the BAL fluid (closed bars) that was not inhibited by 3E10 mAb (open bars) .
  • Data are shown as the mean + sem of 5-6 animals.
  • FIG. 27A Analysis of the number of eosinophils in the BAL
  • FIG. 27B analysis of the number of lymphocytes in the BAL.
  • Lymphocytes were stained with biotinylated clonotypic TCR mAb KJ126 (Cohn, L. et al., 1997 J. Exp. Med. 186:1737-1747) followed by strepavidin-FITC and CD4-PE (Pharmingen, San Diego) .
  • FIG. 28 Inhibition of airway hyperresponsiveness by anti-103 gene product mAb.
  • OVA exposure in TH2 recipient mice resulted in airway hyperresponsiveness (closed squares) compared to mice exposed to PBS (closed circles) .
  • Pretreatment with 103 gene product mAb inhibited OVA induced BHR by 80% (open diamonds) .
  • FIG. 29A-29B Administration of 3E10 mAb or the 103/Ig fusion results in significant decrease in hallmark symptoms of asthma.
  • FIG. 29A Animals were treated with the anti-103 3E10 antibody (listed in the figure as "3E10 mAB”) . As a negative control, a set of animals was treated with a nonspecific rat Ig antibody preparation.
  • FIG. 29B Animals were treated with 103/Ig fusion protein (listed in the figure as "Ig Fus. Prot.") as a negative control, a control set of animals were treated with a non-specific human IgG antibody preparation.
  • Fig. 30 Crosslinking of 103 gene product augments IL-4 and IL-5 cytokine secretion.
  • TH2 effector cells were activated with plate-bound CD3 (3 ⁇ g/ml, 2C11) and CD28 (37.51, 4 ⁇ g/ml, Pharmingen San Diego) and 3E10 (20 ⁇ g/ml) for 48 hrs.
  • IL-4 and IL-5 levels were measured in the supernatant by ELISA.
  • Fig. 31 Renal histology at 72 hours reperfusion
  • Fig. 31A shows a section of untreated mouse kidney tissue
  • Fig. 3 IB shows a section of mouse kidney tissue treated with 200 gene antibody 24 hours prior to, and at 24 hour intervals after the induction of ischemic kidney injury.
  • Fig. 32 Histological scoring of gene 200 blockage in treated (+a200) and untreated (+Rtlg) mouse kidney tissue during renal ischemia/reperfusion injury (RI) , and in non ischemic controls (S) .
  • TH cell subpopulation-related disorders including, but not limited to, atopic conditions, such as asthma and allergy, including allergic rhinitis, psoriasis, the effects of pathogen infection, chronic inflammatory diseases, organ-specific autoimmunity, graft rejection and graft versus host disease, are described.
  • atopic conditions such as asthma and allergy, including allergic rhinitis, psoriasis, the effects of pathogen infection, chronic inflammatory diseases, organ-specific autoimmunity, graft rejection and graft versus host disease.
  • the methods and compositions described herein can also be used to treat ischemic disorders and injuries, including but not limited to, ischemic renal disease and injury, myocardial ischemia such as angina pectoris, as well as ischemic injury to other tissues, including the brain (as in a stroke) , spleen, intestine, lung, and testes.
  • the methods and compositions described herein can also be used to regulate ischemic injury to other types of tissue, such as tumor tissue including, but not limited to tumors of the ovary and uterus.
  • the invention is based, in part, on the evaluation of the expression and role of all genes that are differentially expressed within and/or among TH cell subpopulations in paradigms that are physiologically relevant to TH-mediated immune response and/or TH-subpopulation related disorders. This permits the definition of disease pathways that are useful both diagnostically and therapeutically .
  • the invention is also based in part on the discovery that the genes and gene products of the invention are also involved in processes related to tissue repair and remodeling after injury, particularly after ischemic injury. Thus, the genes and gene products of the invention can also be used to successfully treat such injuries and related disorders.
  • Target genes and/or “fingerprint genes” which are differentially expressed within and among TH cells and TH cell subpopulations in normal and/or disease states, and/or during the differentiation into such mature subpopulations are described in Section 5.4.
  • genes termed “pathway genes”, whose gene products exhibit an ability to interact with gene products involved in TH cell subpopulation-related disorders and/or with gene products which are involved in the differentiation and effector function of the subpopulations are described in Section 5.4.
  • Pathway genes can additionally have fingerprint and/or target gene characteristics. Methods for the identification of such fingerprint, target, and pathway genes are also described in Sections 5.1 and 5.2.
  • differentially expressed genes which are involved in immune disorders, e.g.. TH cell subpopulation-related disorders, and/or which are involved in the differentiation, maintenance and effector function of the subpopulations.
  • immune disorders e.g.. TH cell subpopulation-related disorders
  • levels at which the differential expression of such genes can be exhibited There exist a number of levels at which the differential expression of such genes can be exhibited.
  • differential expression can occur in undifferentiated TH cells versus differentiated or differentiating TH cells (although not necessarily within one TH cell subpopulation versus another) , in naive TH cells versus memory TH cells, within one TH cell subpopulation versus another (e.g.
  • differentially expressed genes can represent target and/or fingerprint genes. Methods for the identification of such differentially expressed genes are described, below, in Section 5.1.1. Methods for the further characterization of such differentially expressed genes, and for their categorization as target and/or fingerprint genes, are presented, below, in Section 5.3.
  • differential expression refers to both quantitative as well as qualitative differences in the genes' temporal and/or cell type expression patterns.
  • a differentially expressed gene can qualitatively have its expression activated or completely inactivated in, for example, normal versus TH cell subpopulation-related disorder states, in one TH cell subpopulation versus another (e.g. , THl versus TH2), in antigen stimulated versus unstimulated sets of TH cells, or in undifferentiated versus differentiated or differentiating TH cells.
  • Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both.
  • a differentially expressed gene can exhibit an expression level which differs, i.e.. is quantitatively increased or decreased, in normal versus TH cell subpopulation-related disorder states, in antigen stimulated versus unstimulated sets of TH cells, in one TH cell subpopulation versus another, or in undifferentiated versus differentiated or differentiating TH cells. Because differentiation is a multistage event, genes which are differentially expressed can also be identified at any such intermediate differentiative stage.
  • the degree to which expression differs need only be large enough to be visualized via standard characterization techniques, such as, for example, the differential display technique described below.
  • standard characterization techniques by which expression differences can be visualized include, but are not limited to, quantitative RT (reverse transcriptase) PCR and Northern analyses and RNase protection techniques.
  • Differentially expressed genes can be further described as target genes and/or fingerprint genes.
  • Fingerprint gene refers to a differentially expressed gene whose expression pattern can be utilized as part of a prognostic or diagnostic TH cell subpopulation-related disorder evaluation, or which, alternatively, can be used in methods for identifying compounds useful for the treatment of TH cell subpopulation-related disorders.
  • a fingerprint gene can also have the characteristics of a target gene or a pathway gene (see below, in Section 5.2).
  • Fingerprint pattern refers to the pattern generated when the expression pattern of a series (which can range from two up to all the fingerprint genes which exist for a given state) of fingerprint genes is determined.
  • a fingerprint pattern can also be used in methods for identifying compounds useful in the treatment of immune disorders, e.g. , by evaluating the effect of the compound on the fingerprint pattern normally displayed in connection with the disease.
  • Target gene refers to a differentially expressed gene involved in TH cell subpopulation-related disorders and/or in differentiation, maintenance and/or effector function of the subpopulations in a manner by which modulation of the level of target gene expression or of target gene product activity can act to ameliorate symptoms of TH cell subpopulation-related disorders. For example, such modulation can result either the depletion or stimulation of one or more TH cell subpopulation, which, in turn, brings about the amelioration of immune disorder, e.g. , TH cell subpopulation disorder, symptoms .
  • immune disorder e.g. , TH cell subpopulation disorder, symptoms .
  • Stimulation can refer to an effective increase in the number of cells belonging to a T cell population, such as a TH cell subpopulation, via, for example, the proliferation of such TH cell subpopulation cells.
  • the term can also refer to an increase in the activity of cells belonging to a TH cell subpopulation, as would by evidenced, for example, by a per cell increase in the expression of the TH cell subpopulation-specific cytokine 5 pattern.
  • Depletion can refer to an effective reduction in the number of cells belonging to a T cell population, such as a TH cell subpopulation, via, for example, a reduction in the proliferation of such TH cell
  • the term can also refer to a decrease in the activity of cells belonging to a TH cell subpopulation, as would be evidenced, for example, by a per cell decrease in the expression of the TH cell subpopulation- specific cytokine pattern.
  • TH cell subpopulation-related disorders include, for example, atopic conditions, such as asthma and allergy, including allergic rhinitis, the effects of pathogen, including viral, infection, chronic inflammatory diseases, psoriasis, glomerular nephritis, organ-specific autoimmunity,
  • a target gene can also have the characteristics of a fingerprint gene and/or a pathway gene (as described, below, in Section 5.2).
  • a variety of methods can be utilized for the identification of genes which are involved in immune disorder states, e.g. , TH cell subpopulation-related disorder states, and/or which are involved in differentiation, maintenance
  • genes which are differentially expressed within and among TH cell subpopulations including but not limited to THl and TH2 subpopulations.
  • Such genes can be involved in, for example,
  • TH cells can be induced to differentiate into either THl or TH2 states, can be stimulated with, for example, a foreign antigen, and can be collected at various points during the procedure for analysis
  • transgenic T cell paradigm In one embodiment of such a paradigm, referred to herein as the "transgenic T cell paradigm", transgenic animals, preferably mice, are utilized which have been engineered to express a particular T cell receptor, such that the
  • T cell population of the immune system of such a transgenic animal recognizes only one antigen.
  • Such a system is preferred in that it provides a source for a large population of identical T cells whose naivete can be assured, and whose response to the single antigen it recognizes is
  • T helper cells isolated from such a transgenic animal are induced, in vitro, to differentiate into TH cell subpopulations such as THl, TH2 , or THO cell subpopulations.
  • TH cell subpopulations such as THl, TH2 , or THO cell subpopulations.
  • one T helper cell group (the THl group) is exposed to IL-12, a cytokine known to induce
  • a second T helper cell group (the TH2 group) is exposed to IL-4, a cytokine known to induce differentiation into the TH2 state, and a third group is allowed, by a lack of cytokine-mediated induction, to enter a TH-undirected state.
  • T cell line paradigm uses mature TH cell clones, such as THl and TH2 and THl-like and TH2-like cell lines, preferably human cell lines.
  • TH cell lines can include, but are not limited to the following well known murine cell lines: Doris, AE7 , D10.G4, DAX, Dl.l and CDC25.
  • T cell lines can be derived from normal individuals as well as individuals exhibiting TH cell subpopulation-related disorders, such as, for example, chronic inflammatory diseases and disorders, such as Crohn ' s disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g.
  • the TH cell clones can be stimulated in a variety of ways. Such stimulation methods include, but are not limited to, pharmacological methods, such as exposure to phorbol esters, calcium ionophores, or lectins (e.g. , Concanavalin A) , by treatment with antibodies directed against T-cell receptor epitopes (e.g. , anti-CD3 antibodies) or exposure, in the presence of an appropriate antigen presenting cell (APC) , to an antigen that the particular TH cells are known to recognize. Following such primary stimulation, the cells can be maintained in culture without stimulation and, for example, in the presence of IL-2 , utilizing standard techniques well known to those of skill in the art. The cells can then be exposed to one or more additional cycles of stimulation and maintenance.
  • pharmacological methods such as exposure to phorbol esters, calcium ionophores, or lectins (e.g. , Concanavalin A)
  • T-cell receptor epitopes e.g. ,
  • a third paradigm referred to herein as an "in vivo paradigm" can also be utilized to discover differentially expressed gene sequences.
  • In vivo stimulation of animal models forms the basis for this paradigm.
  • the in vivo nature of the stimulation can prove to be especially predictive of the analogous responses in living patients.
  • Stimulation can be accomplished via a variety of methods. For example, animals, such as transgenic animals described earlier in this Section, can be injected with appropriate antigen and appropriate cytokine to drive the desired TH cell differentiation. Draining lymph nodes can then be harvested at various time points after stimulation. Lymph nodes from, for example, THl-directed animals can be compared to those of TH2-directed animals.
  • animal models representing both models of normal immune differentiation and function as well as those representing immune disorders can be utilized for this in vivo paradigm.
  • Cell samples can be collected during any point of such a procedure. For example, cells can be obtained following any stimulation period and/or any maintenance period. Additionally, cells can be collected during various points during the TH cell differentiation process. RNA collected from such samples can be compared and analyzed according to, for example, methods described, below, in Section 5.1.1.2. For example, RNA from THO, THl and TH2 groups isolated at a given time point can then be analyzed and compared. Additionally, RNA from stimulated and non-stimulated cells within a given TH cell group can also be compared and analyzed. Further, RNA collected from undifferentiated TH cells can be compared to RNA collected from cells at various stages during the differentiative process which ultimately yields TH cell subpopulations.
  • RNA either total or mRNA
  • RNA isolation technique which does not select against the isolation of mRNA can be utilized for the purification of such RNA samples. See, for example, Ausubel, F.M. et al. , eds., 1987-1993, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York, which is incorporated herein by reference in its entirety.
  • RNA samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, P. (1989, U.S. Patent No. 4,843,155), which is incorporated herein by reference in its entirety.
  • Transcripts within the collected RNA samples which represent RNA produced by differentially expressed genes can be identified by utilizing a variety of methods which are well known to those of skill in the art. For example, differential screening (Tedder, T.F. et al. , 1988, Proc. Natl. Acad. Sci. USA 85:208-212), subtractive hybridization (Hedrick, S.M. et al..
  • differential display Liang, P. and Pardee, A.B., 1992, Science 257:967-971; U.S. Patent No. 5,262,311, which are incorporated herein by reference in their entirety
  • differential display can be utilized to identify nucleic acid sequences derived from genes that are differentially expressed.
  • Differential screening involves the duplicate screening of a cDNA library in which one copy of the library is screened with a total cell cDNA probe corresponding to the mRNA population of one cell type while a duplicate copy of the cDNA library is screened with a total cDNA probe corresponding to the mRNA population of a second cell type.
  • one cDNA probe can correspond to a total cell cDNA probe of a cell type or tissue derived from a control subject
  • the second cDNA probe can correspond to a total cell cDNA probe of the same cell type or tissue derived from an experimental subject.
  • Those clones which hybridize to one probe but not to the other potentially represent clones derived from genes differentially expressed in the cell type of interest in control versus experimental subjects.
  • Subtractive hybridization techniques generally involve the isolation of mRNA taken from two different sources, the hybridization of the mRNA or single-stranded cDNA reverse- transcribed from the isolated mRNA, and the removal of all hybridized, and therefore double-stranded, sequences.
  • the remaining non-hybridized, single-stranded cDNAs potentially represent clones derived from genes that are differentially expressed among the two mRNA sources.
  • Such single-stranded cDNAs are then used as the starting material for the construction of a library comprising clones derived from differentially expressed genes.
  • the differential display technique is a procedure, utilizing the well-known polymerase chain reaction (PCR; the experimental embodiment set forth in Mullis, K.B., 1987, U.S. Patent No. 4,683,202), which allows for the identification of sequences derived from genes which are differentially expressed.
  • PCR polymerase chain reaction
  • isolated RNA is reverse-transcribed into single-stranded cDNA, utilizing standard techniques which are well known to those of skill in the art.
  • Primers for the reverse transcriptase reaction can include, but are not limited to, oligo dT-containing primers, preferably of the 3' primer type of oligonucleotide described below.
  • this technique uses pairs of PCR primers, as described below, which allow for the amplification of clones representing a reproducible subset of the RNA transcripts present within any given cell. Utilizing different pairs of primers allows each of the primed mRNA transcripts present in a cell to be amplified. Among such amplified transcripts can be identified those which have been produced from differentially expressed genes.
  • the 3 ' oligonucleotide primer of the primer pairs can contain an oligo dT stretch of 10-13, preferably 11, dT nucleotides at its 5' end, which hybridizes to the poly (A) tail of mRNA or to the complement of a cDNA reverse transcribed from an mRNA poly (A) tail.
  • the primer can contain one or more, preferably two, additional nucleotides at its 3' end.
  • the additional nucleotides allow the primers to amplify only a subset of the mRNA derived sequences present in the sample of interest. This is preferred in that it allows more accurate and complete visualization and characterization of each of the bands representing amplified sequences.
  • the 5 ' primer can contain a nucleotide sequence expected, statistically, to have the ability to hybridize to cDNA sequences derived from the cells or tissues of interest.
  • the nucleotide sequence can be an arbitrary one, and the length of the 5 ' oligonucleotide primer can range from about 9 to about 15 nucleotides, with about 13 nucleotides being preferred.
  • Arbitrary primer sequences cause the lengths of the amplified partial cDNAs produced to be variable, thus allowing different clones to be separated by using standard denaturing sequencing gel electrophoresis.
  • PCR reaction conditions should be chosen which optimize amplified product yield and specificity, and, additionally, produce amplified products of lengths which can be resolved utilizing standard gel electrophoresis techniques.
  • reaction conditions are well known to those of skill in the art, and important reaction parameters include, for example, length and nucleotide sequence of oligonucleotide primers as discussed above, and annealing and elongation step temperatures and reaction times.
  • the pattern of clones resulting from the reverse transcription and amplification of the mRNA of two different cell types is displayed via sequencing gel electrophoresis and compared. Differentially expressed genes are indicated by differences in the two banding patterns.
  • differential expression of such putatively differentially expressed genes should be corroborated. Corroboration can be accomplished via, for example, such well known techniques as Northern analysis, quantitative RT/PCR, or RNAse protection.
  • the differentially expressed genes can be further characterized, and can be identified as target and/or fingerprint genes, as discussed, below, in Section 5.3.
  • the amplified sequences of differentially expressed genes obtained through, for example, differential display can be used to isolate full length clones of the corresponding gene.
  • the full length coding portion of the gene can readily be isolated, without undue experimentation, by molecular biological techniques well known in the art.
  • the isolated differentially expressed amplified fragment can be labeled and used to screen a cDNA library.
  • the labeled fragment can be used to screen a genomic library.
  • PCR technology can also be utilized to isolate full length cDNA sequences.
  • the isolated, amplified gene fragments obtained through differential display have 5' terminal ends at some random point within the gene and usually have 3 ' terminal ends at a position corresponding to the 3 ' end of the transcribed portion of the gene.
  • the remainder of the gene i.e. , the 5' end of the gene, when utilizing differential display
  • RNA can be isolated, following standard procedures, from an appropriate tissue or cellular source.
  • a reverse transcription reaction can then be performed on the RNA using an oligonucleotide primer complimentary to the mRNA that corresponds to the amplified fragment, for the priming of first strand synthesis. Because the primer is anti-parallel to the mRNA, extension will proceed toward the 5' end of the mRNA.
  • the resulting RNA/DNA hybrid can then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid can be digested with RNAase H, and second strand synthesis can then be primed with a poly-C primer.
  • pathway gene refers to a gene whose gene product exhibits the ability to interact with gene products involved in TH cell subpopulation-related disorders and/or to interact with gene products which are involved in differentiation, maintenance and/or effector function of TH cell subpopulations.
  • a pathway gene can be differentially expressed and, therefore, can have the characteristics of a target and/or fingerprint gene, as described, above, in Section 5.1.
  • Any method suitable for detecting protein-protein interactions can be employed for identifying pathway gene products by identifying interactions between gene products and gene products known to be involved in TH cell subpopulation-related disorders and/or involved in differentiation, maintenance, and/or effector function of the subpopulations.
  • Such known gene products can be cellular or extracellular proteins.
  • Those gene products which interact with such known gene products represent pathway gene products and the genes which encode them represent pathway genes.
  • a pathway gene product can be used, in conjunction with standard techniques, to identify its corresponding pathway gene. For example, at least a portion of the amino acid sequence of the pathway gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g. , Creighton, 1983, "Proteins: Structures and Molecular Principles", W.H. Freeman & Co., N.Y., pp.34-49).
  • the amino acid sequence obtained can be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for pathway gene sequences. Screening can be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and for screening are well-known. (See, e.g. , Ausubel, supra. , and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al., eds. Academic Press, Inc., New York).
  • methods can be employed which result in the simultaneous identification of pathway genes which encode proteins interacting with a protein involved in TH cell subpopulation-related disorder states and/or differentiation, maintenance, and/or effector function of the subpopulations.
  • methods include, for example, probing expression libraries with labeled protein known or suggested to be involved in the disorders and/or the differentiation, maintenance, and/or effector function of the subpopulations, using this protein in a manner similar to the well known technique of antibody probing of ⁇ gtll libraries.
  • plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, in this case, a protein known to be involved in TH cell subpopulation differentiation or effector function, or in TH cell subpopulation-related disorders, and the other consists of the activator protein's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombined into this plasmid as part of a cDNA library.
  • the plasmids are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding sites.
  • a reporter gene e.g., lacZ
  • the two-hybrid system or related methodology can be used to screen activation domain libraries for proteins that interact with a known "bait" gene product.
  • gene products known to be involved in TH cell subpopulation-related disorders and/or differentiation, maintenance, and/or effector function of the subpopulations can be used as the bait gene products.
  • Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
  • This library and a plasmid encoding a hybrid of the bait gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene.
  • the bait gene can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA- binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.
  • a cDNA library of the cell line from which proteins that interact with bait gene product are to be detected can be made using methods routinely practiced in the art.
  • the cDNA fragments can be inserted into a vector such that they are translationally fused to the activation domain of GAL4.
  • This library can be co-transformed along with the bait gene- GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence.
  • a cDNA encoded protein, fused to GAL4 activation domain, that interacts with bait gene product will reconstitute an active GAL4 protein and thereby drive expression of the lacZ gene.
  • Colonies which express lacZ can be detected by their blue color in the presence of X-gal.
  • the cDNA can then be purified from these strains, and used to produce and isolate the bait gene-interacting protein using techniques routinely practiced in the art.
  • pathway gene Once a pathway gene has been identified and isolated, it can be further characterized as, for example, discussed below, in Section 5.3.
  • Differentially expressed genes such as those identified via the methods discussed, above, in Section 5.1
  • pathway genes such as those identified via the methods discussed, above, in Section 5.2, above, as well as genes identified by alternative means
  • genes identified by alternative means can be further characterized by utilizing, for example, methods such as those discussed herein.
  • Such genes will be referred to herein as "identified genes”. Analyses such as those described herein yield information regarding the biological function of the identified genes. An assessment of the biological function of the differentially expressed genes, in addition, will allow for their designation as target and/or fingerprint genes.
  • any of the differentially expressed genes whose further characterization indicates that a modulation of the gene's expression or a modulation of the gene product's activity can ameliorate any of the TH cell subpopulation- related disorders of interest will be designated "target genes", as defined, above, in Section 5.1.
  • target genes and target gene products along with those discussed below, will constitute the focus of the compound discovery strategies discussed, below, in Section 5.8.
  • target genes, target gene products and/or modulating compounds can be used as part of the TH cell subpopulation- disorder treatment methods described, below, in Section 5.9.
  • Such methods can include, for example, methods whereby the TH cell subpopulation of interest is selectively depleted or repressed, or, alternatively, stimulated or augmented.
  • any of the differentially expressed genes whose further characterization indicates that such modulations can not positively affect TH cell subpopulation-related disorders of interest, but whose expression pattern contributes to a gene expression "fingerprint” pattern correlative of, for example, a THl/TH2-related disorder state, will be designated a "fingerprint gene”. "Fingerprint patterns" will be more fully discussed, below, in Section 5.12.1. It should be noted that each of the target genes can also function as fingerprint genes, as well as can all or a portion of the pathway genes.
  • pathway genes can also be characterized according to techniques such as those described herein. Those pathway genes which yield information indicating that modulation of the gene's expression or a modulation of the gene product's activity can ameliorate any a TH cell subpopulation-related disorder will also be designated "target genes". Such target genes and target gene products, along with those discussed above, will constitute the focus of the compound discovery strategies discussed, below, in Section 5.8 and can be used as part of the treatment methods described in Section 5.9, below.
  • pathway gene's characterization indicates that modulation of gene expression or gene product activity can not positively affect TH cell subpopulation- related disorders of interest, but whose expression is differentially expressed and contributes to a gene expression fingerprint pattern correlative of, for example, a TH1/TH2- related disorder state
  • pathway genes can additionally be designated as fingerprint genes.
  • nucleotide sequence of the identified genes which can be obtained by utilizing standard techniques well known to those of skill in the art, can, for example, be used to reveal homologies to one or more known sequence motifs which can yield information regarding the biological function of the identified gene product .
  • an analysis of the tissue and/or cell type distribution of the mRNA produced by the identified genes can be conducted, utilizing standard techniques well known to those of skill in the art.
  • Such techniques can include, for example, Northern, RNAse protection, and RT-PCR analyses.
  • analyses provide information as to, for example, whether the identified genes are expressed in cell types expected to contribute to the specific TH cell subpopulation-related disorders of interest.
  • analyses can also provide quantitative information regarding steady state mRNA regulation, yielding data concerning which of the identified genes exhibits a high level of regulation in cell types which can be expected to contribute to the TH cell subpopulation- related disorders of interest.
  • standard in situ hybridization techniques can be utilized to provide information regarding which cells within a given tissue or population of cells express the identified gene.
  • Such an analyses can provide information regarding the biological function of an identified gene relative to a given TH cell subpopulation-related disorder in instances wherein only a subset of the cells within a tissue or a population of cells is thought to be relevant to the disorder.
  • the sequences of the identified genes can be used, utilizing standard techniques, to place the genes onto genetic maps, e.g. , mouse (Copeland, N.G. and Jenkins, N.A., 1991, Trends in Genetics 7:113-118) and human genetic maps (Cohen, D. , et al., 1993, Nature 366: 698-701) .
  • genetic maps e.g. , mouse (Copeland, N.G. and Jenkins, N.A., 1991, Trends in Genetics 7:113-118) and human genetic maps (Cohen, D. , et al., 1993, Nature 366: 698-701) .
  • Such mapping information can yield information regarding the genes' importance to human disease by, for example, identifying genes which map within genetic regions to which known genetic TH cell subpopulation-related disorders map.
  • Such regions include, for example, the mouse Scl-1 locus, which is suspected to be involved in Leishmaniasis, or the human 5q31.1 chromosomal region which contains one or more loci thought to regulate IgE production in a nonantigen-specific fashion, and can, therefore, be involved in allergy, a TH2- like-related disorder (Marsh, D. et al., 1994, Science 264:1152-1156) .
  • the biological function of the identified genes can be more directly assessed by utilizing relevant in vivo and in vitro systems.
  • In vivo systems can include, but are not limited to, animal systems which naturally exhibit the symptoms of immune disorders, or ones which have been engineered to exhibit such symptoms.
  • Such systems can include systems for the further characterization of the cell type differentiation and effector function, and can include, but are not limited to transgenic animal systems such as those described, above, in Section 5.1.1.1, and Section 5.7.1, below.
  • In vitro systems can include, but are not limited to, cell-based systems comprising, for example, THl or TH2 cell types.
  • the TH subpopulation cells can be wild type cells, or can be non-wild type cells containing modifications known or suspected of contributing to the TH cell subpopulation-related disorder of interest. Such- systems are discussed in detail, below, in Section 5.7.2.
  • the expression of these genes can be modulated within the in vivo and/or in vitro systems, i.e.
  • the activity of the product of the identified gene can be modulated by either increasing or decreasing the level of activity in the in vivo and/or in vitro system of interest, and its subsequent effect then assayed.
  • relevant treatment can include not only a modulation of gene expression and/or gene product activity, but can also include a selective depletion or stimulation of the TH cell subpopulation of interest. Characterization procedures such as those described herein can indicate where such modulation should be positive or negative. As used herein, "positive modulation” refers to an increase in gene expression or activity of the gene or gene product of interest, or to a stimulation of a TH cell subpopulation, relative to that observed in the absence of the modulatory treatment.
  • “Negative modulation”, as used herein, refers to a decrease in gene expression or activity, or a depletion of a TH cell subpopulation, relative to that observed in the absence of the modulatory treatment.
  • “Stimulation” and “depletion” are as defined, above, in Section 3. Methods of treatment are discussed, below, in Section 5.9.
  • differentially expressed and pathway genes of the invention are listed below, in Table 1. Differentially expressed gene sequences are shown in FIGS. 2, 4A, 9 and 12- 15, 17, 22 and 24.
  • the nucleotide sequences identified via differential display analysis are referred to herein as band 10, 54, 57, 102, 103, 105, 106, 161 and 200.
  • the genes corresponding to these sequences are referred to herein as the 10, 54, 57, 102, 103, 106, 161 and 200 genes, respectively.
  • Table 1 lists differentially expressed genes identified through, for example, the paradigms discussed, above, in Section 5.1.1.1, and below, in the Examples presented in Sections 6-8.
  • Table 1 summarizes information regarding the further characterization of such genes.
  • Table 2 lists E. coli clones, deposited with the Agricultural Research Service Culture Collection (NRRL) or the American Type Culture Collection (ATCC) , which contain sequences found within the genes of Table 1.
  • TH Inducible refers to those cases where differential expression arises upon exposure of the cell type of interest to an agent capable of bringing about TH cell stimulation or activation.
  • THl under this column, refers to a sequence corresponding to a gene expressed preferentially in mature, fully differentiated THl cells relative to TH2 cells.
  • TH2 under this column, refers to a sequence corresponding to a gene preferentially expressed in mature, fully differentiated TH2 cell subpopulations relative to THl cell subpopulations. Preferential expression can be qualitative or quantitative, as described, above, in Section 5.1. Tissue expression patterns are also summarized in Table 1. The column headed "Tissue/Cell Dist.” lists tissues and/or cell types in which expression of the gene has been tested and whether expression of the gene within a given tissue or cell type has been observed.
  • the physical locus to which the gene maps on the human and/or mouse chromosome map is indicated in the column headed "Locus".
  • references i.e. , citations and/or gene names
  • the genes listed in Table 1 can be obtained using cloning methods well known to those of skill in the art, and include but are not limited to the use of appropriate probes to detect the genes within an appropriate cDNA or gDNA (genomic DNA) library.
  • Probes for the sequences reported herein can be obtained directly from the isolated clones deposited with the NRRL, as indicated in Table 2, below.
  • oligonucleotide probes for the genes can be synthesized based on the DNA sequences disclosed herein in FIGS. 2, 4A, 9, 12-15, 17, 22 and 24.
  • synthetic oligonucleotides can be synthesized or produced based on the sequences provided for the previously known genes described in the following references: granzyme A, Hanukah factor: Masson, D.
  • the probes can be used to screen cDNA libraries prepared from an appropriate cell or cell line in which the gene is transcribed.
  • Appropriate cell lines can include, for example, Dorris, AE7 , D10.G4, DAX, Dl.l and CDC25 cell lines.
  • purified primary naive T cells derived from either transgenic or non-transgenic strains can be used.
  • genes described herein can be cloned from a cDNA library constructed from, for example, NIH 3T3 cell lines stably transfected with the Ha-ras(EJ) gene, 5C10 cells, and peripheral blood lymphocytes.
  • Table 2 lists isolated E. coli clones which contain sequences within the novel genes listed in Table 1.
  • differentiated gene i.e. target and fingerprint gene
  • pathway gene refers to (a) a gene containing: at least one of the DNA sequences and/or fragments thereof that are disclosed herein (as shown in FIGS. 2, 4A, 9, 12-15, 17, 22 and 24), or contained in the clones listed in Table 2, as deposited with the NRRL or ATCC; (b) any DNA sequence or fragment thereof that encodes the amino acid sequence encoded by: the DNA sequences disclosed herein (as shown in FIGS.
  • the nucleic acid molecules that nybridize to the complements of the DNA sequences disclosed herein encode gene products, e.g., gene products that are functionally equivalent to a gene product encoded by a gene of (a) , above.
  • gene products e.g., gene products that are functionally equivalent to a gene product encoded by a gene of (a) , above.
  • “Functionally equivalent”, as utilized herein, refers to a protein capable of exhibiting a substantially similar in vivo activity as the endogenous differentially expressed or pathway gene products encoded by the differentially expressed or pathway gene sequences described in Section 5.4, above.
  • “functionally equivalent” can refer to peptides capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the endogenous differentially expressed or pathway gene product would.
  • Functionally equivalent gene products therefore include naturally occurring differentially expressed or pathway gene products present in the same or different species.
  • Functionally equivalent differentially expressed and/or pathway gene products also include gene products that retain at least one of the biological activities of the differentially expressed and/or pathway gene products described above; e.g., which are encoded by the coding sequences disclosed herein (as shown in FIGS.
  • the functionally equivalent gene products of the pathway and/or differentially expressed genes of the invention also include gene products which are recognized by and bind to antibodies (polyclonal or monoclonal) directed against the differentially expressed and/or pathway gene products described above; e.g., which are encoded by the coding sequences disclosed herein (as shown in FIGS. 2, 4A, 9, 12-15, 17, 22 and 24), contained in clones listed in Table 2, as deposited with the NRRL or ATCC.
  • the invention also includes degenerate variants of sequences (a) through (d) .
  • the invention encompasses the following nucleotides, host cells expressing such nucleotides and the expression products of such nucleotides: (a) nucleotides that encode a mammalian differentially expressed and/or pathway gene product including, but not limited to a human and murine 10, 54, 57, 105, 106, 161 and 200 gene product; (b) nucleotides that encode portions of differentially expressed and/or pathway gene product that corresponds to its functional domains, and the polypeptide products encoded by such nucleotide sequences, and in which, in the case of receptor- type gene products, such domains include, but are not limited to extracellular domains (ECD) , transmembrane domains (TM) and cytoplasmic domains (CD) ; (c) nucleotides that encode mutants of a differentially expressed and/or pathway gene, product, in which all or part of one of its domains is deleted or altered, and which, in the case of receptor-type gene products, such mutants include
  • nucleotide sequences of the invention further include nucleotide sequences corresponding to the nucleotide sequences of (a) -(d) above wherein one or more of the exons, or fragments thereof, have been deleted.
  • nucleotide sequences of the invention still further include nucleotide sequences that have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more nucleotide sequence identitiy to the nucleotide sequences of (a) -(d) above.
  • the nucleotide sequences of the invention also include nucleotide sequences that encode polypeptides having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or higher amino acid sequence identity to the polypeptides encoded by the nucleotide sequences of (a) -(d) above.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino aor nucleic acid sequence) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc . Natl . Acad . Sci . U . S . A 87 :2264-2268 , modified as in Karlin and Altschul (1993) Proc . Natl . Acad . Sci . U . S . A .
  • Gapped BLAST can be utilized as described in Altschul et al . , 1997, Nucleic Acids Res . 25:3389-3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id . ) .
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • the default parameters of the respective programs can be used (see, e.g., http: / /www. ncbi. nlm.nih.gov) .
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4 : 11-11 . Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • the invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the DNA sequences (a) through (d) , in the preceding paragraph.
  • nucleic acid molecules of the invention that hybridize to the above described DNA sequences include oligodeoxyoligonucleotides ("oligos") which hybridize under highly stringent or stringent conditions to the DNA sequences (a) through (d) in the preceding paragraph.
  • hybridization is carried out at about 20-25 degrees below Tm (for DNA-DNA hybrids) or about 10-15 degrees below Tm (for RNA-DNA hybrids) .
  • Other examplary highly stringent conditions may refer, e.g., to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos) , 48°C (for 17-base oligos) , 55°C (for 20-base oligos) , and 60°C (for 23-base oligos) .
  • nucleic acid molecules can encode or act as target gene antisense molecules, useful, for example, in target gene regulation and/or as antisense primers in amplification reactions of target, fingerprint, and/or pathway gene nucleic acid sequences. Further, such sequences can be used as part of ribozyme and/or triple helix sequences, also useful for target gene regulation. Still further, such molecules can be used as components of diagnostic methods whereby the presence of, or predisposition to, an immune disorder, e.g. , TH cell subpopulation-related disorder, can be detected.
  • an immune disorder e.g. , TH cell subpopulation-related disorder
  • Fragments of the differentially expressed and pathway genes of the invention can be at least 10 nucleotides in length.
  • the fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more contiguous nucleotides in length.
  • the fragments can comprise sequences that encode at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or more contiguous amino acid residues of the differentially express and pathway gene products.
  • Fragments of the differentially expressed and pathway nucleic acid molecules of the invention can also refer to exons or introns of the above described nucleic acid molecules, as well as portions of the coding regions of such nucleic acid molecules that encode function domains such as extracellular domains (ECD) , transmembrane domains (TM) and cytoplasmic domains (CD) .
  • ECD extracellular domains
  • TM transmembrane domains
  • CD cytoplasmic domains
  • the invention also encompasses (a) DNA vectors that contain any of the foregoing coding sequences and/or their complements (i.e. , antisense) ; (b) DNA expression vectors that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell.
  • regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
  • Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast ⁇ -mating factors.
  • the invention includes fragments of any of the DNA sequences disclosed herein.
  • homologs of these gene sequences and/or full length coding sequences of these genes can be identified and isolated, without undue experimentation, by molecular biological techniques well known in the art. Further, there can exist genes at other genetic loci within the genome of the same species that encode proteins which have extensive homology to one or more domains of such gene products. These genes can also be identified via similar techniques.
  • the isolated differentially expressed gene sequence can be labeled and used to screen a cDNA library constructed from mRNA obtained from the organism of interest.
  • Hybridization conditions should be of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled sequence was derived.
  • cDNA screening can also identify clones derived from alternatively spliced transcripts in the same or different species.
  • the labeled fragment can be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • Low stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived.
  • a previously unknown differentially expressed or pathway gene-type sequence can be isolated by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the gene of interest.
  • the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from human or non-human cell lines or tissue known or suspected to express a differentially expressed or pathway gene allele.
  • the PCR product can be subcloned and sequenced to insure that the amplified sequences represent the sequences of a differentially expressed or pathway gene-like nucleic acid sequence.
  • the PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods.
  • the amplified fragment can be used to screen a bacteriophage cDNA library.
  • the labeled fragment can be used to screen a genomic library.
  • RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source.
  • a reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5 ' end of the amplified fragment for the priming of first strand synthesis.
  • the resulting RNA/DNA hybrid can then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid can be digested with RNAase H, and second strand synthesis can then be primed with a poly-C primer.
  • cDNA sequences upstream of the amplified fragment can easily be isolated.
  • DNA sequence polymorphisms of a differentially expressed or pathway gene identified by the methods of the present invention will exist within a population of individual organisms (e.g., within a human population). Such polymorphisms may exist, for example, among individuals within a population due to natural allelic variation. Such polymorphisms include ones that lead to changes in amino acid sequence.
  • An allele is one of a group of genes which occurs alternatively at a given genetic locus.
  • an "allelic variant” refers to a nucleotide sequence which occurs at a given locus or to a gene product encoded by the nucleotide sequence. Natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
  • allelic variants can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals.
  • gene and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. The term can further include nucleic acid molecules comprising upstream and/or exon/intron sequences and structure.
  • allelic variant of the differentially expressed and pathway genes and gene products of the present invention any and all nucleotide variations and/or amino acid polymorphisms or variations that are the result of natural allelic variation of the differentially expressed pathway genes and/or gene products are intended to be within the scope of the present invention.
  • allelic variants include, but are not limited to, ones that do not alter the functional activity of a differentially expressed or pathway gene product of the invention.
  • Variants also include, but are not limited to "mutant alleles.”
  • a "mutant allele" of a differentially expressed or pathway gene or gene product of the invention is an allelic variant which does alter the functional activity of the differentially expressed or pathway gene product encoded by that gene.
  • the differentially expressed or pathway gene identified is the normal, or wild type, gene
  • this gene can be used to isolate mutant alleles of the gene. Such an isolation is preferable in processes and disorders which are known or suspected to have a genetic basis. Mutant alleles can be isolated from individuals either known or suspected to have a genotype which contributes to TH cell subpopulation- disorder related symptoms. Mutant alleles and mutant allele products can then be utilized in the therapeutic and diagnostic assay systems described below.
  • a cDNA of a mutant gene can be isolated, for example, by using PCR, a technique which is well known to those of skill in the art.
  • the first cDNA strand can be synthesized by hybridizing a oligo-dT oligonucleotide to mRNA isolated from tissue known to, or suspected of, being expressed in an individual putatively carrying the mutant allele, and by extending the new strand with reverse transcriptase.
  • the second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene.
  • the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art.
  • DNA sequence analysis By comparing the DNA sequence of the mutant gene to that of the normal gene, the mutation (s) responsible for the loss or alteration of function of the mutant gene product can be ascertained.
  • a genomic or cDNA library can be constructed and screened using DNA or RNA, respectively, from a tissue known to or suspected of expressing the gene of interest in an individual suspected of or known to carry the mutant allele.
  • the normal gene or any suitable fragment thereof can then be labeled and used as a probed to identify the corresponding mutant allele in the library.
  • the clone containing this gene can then be purified through methods routinely practiced in the art, and subjected to sequence analysis as described, above, in this Section.
  • an expression library can be constructed utilizing DNA isolated from or cDNA synthesized from a tissue known to or suspected of expressing the gene of interest in an individual suspected of or known to carry the mutant allele.
  • gene products made by the putatively mutant tissue can be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal gene product, as described, below, in Section 5.6.
  • For screening techniques see, for example, Harlow, E. and Lane, eds . , 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.)
  • the mutation results in an expressed gene product with altered function e.g.
  • allelic variants and/or mutant variants of the differentially expressed and pathway genes of the invention include single nucleotide polymorphisms (SNPs) , including biallelic SNPs or biallelic markers which have two alleles, both of which are present at a fairly high frequency in a population of organisms.
  • Conventional techniques for detecting SNPs include, e.g., conventional dot blot analysis, single stranded conformational polymorphism (SSCP) analysis (see, e.g., Orita et al . , 1989, Proc . Natl . Acad . Sci . USA 86:2766-2770) , denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other routine techniques well known in the art (see, e.g., Sheffield et al . , 1989, Proc . Natl . Acad . Sci . 86 : 5855- 5892; Grompe, 1993, Nature Genetics 5:111-117).
  • SSCP single stranded conformational polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • heteroduplex analysis mismatch cleavage detection
  • other routine techniques well known in the art see, e.g., Sheffield
  • detecting and mapping SNPs involve microsequencing techniques wherein an SNP site in a target DNA is detected by a single nucleotide primer extension reaction (see, e.g., Goelet et al . , PCT Publication No. WO 92/15712; Mundy, U.S. Patent No. 4,656,127; Vary and Diamond, U.S. Patent No. 4,851,331; Cohen et al . , PCT Publication No. WO 91/02087; Chee et al . , PCT Publication No. Wo 95/11995; Landegren et al . , 1988, Science 241 : 1077- 1080; Nicerson et al .
  • Differentially expressed and pathway gene products include those proteins encoded by the differentially expressed and pathway genes corresponding to the gene sequences described in Section 5.4, above, as, for example, the peptides listed in FIGS. 9, 17, 22 and 24.
  • differentially expressed and pathway gene products can include proteins that represent functionally equivalent gene products.
  • gene products include, but are not limited to natural variants of the peptides listed in FIGS. 9, 17, 22 and 24.
  • Such an equivalent differentially expressed or pathway gene product can contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the differentially expressed or pathway gene sequences described, above, in Section 5.4, but which result in a silent change, thus producing a functionally equivalent differentially expressed or pathway gene product.
  • Amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Functionally equivalent” refers to a protein capable of exhibiting a substantially similar in vivo activity as the endogenous differentially expressed or pathway gene products encoded by the differentially expressed or pathway gene sequences described in Section 5.4, above.
  • “functionally equivalent” can refer to peptides capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the endogenous differentially expressed or pathway gene product would.
  • Peptides corresponding to one or more domains of the differentially expressed or pathway gene products e.g.
  • differentially expressed or pathway gene products e.g. , in the case of receptor-type gene products, proteins in which the full length differentially expressed or pathway gene products, a differentially expressed or pathway gene peptide or truncated differentially expressed or pathway gene product is fused to an unrelated protein are also within the scope of the invention and can be designed on the basis of the differentially expressd or pathway gene nucleotide and amino acid sequences disclosed in this Section and in Section 5.4, above.
  • Such fusion proteins include but are not limited to IgFC fusions which stabilize the differentially expressed or pathway gene and prolong half-life in vivo; or fusions to any amino acid sequence that allows the fusion protein to be anchored to the cell membrane, allowing peptides to be exhibited on the cell surface; or fusions to an enzyme, fluorescent protein, or luminescent, protein which provide a marker function.
  • Other mutations to the differentially expressed or pathway gene product coding sequence can be made to generate polypeptides that are better suited for expression, scale up, etc. in the host cells chosen.
  • cysteine residues can be deleted or substituted with another amino acid in order to eliminate disulfide bridges; in the case of secreted or transmembrane proteins, N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
  • N-X-S or N-X-T glycosylation recognition sequences
  • an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the protein at the modified tripeptide sequence.
  • the differentially expressed or pathway gene products of the invention comprise at least as many continguous amino acid residues as necessary to represent an epitope fragment (that is to be recognized by an antibody directed to the differentially expressed or pathway gene product) .
  • such protein fragments or peptides can comprise at least about 8 contiguous amino acid residues from a full length differentially expressed or pathway gene product.
  • the protein fragments and peptides of the invention can comprise about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or more contiguous amino acid residues of a differentially express or pathway gene product.
  • Peptides and/or proteins corresponding to one or more domains of the differentially expressed or pathway gene products are also within the scope of the present invention.
  • fusion proteins in which a differentially expressed or pathway gene product, or a portion thereof such as a truncated differentially expressed or pathway gene product or a domain of a differentially expressed or pathway gene product, is fused to an unrelated protein are within the scope of the present invention.
  • Such proteins and peptides can be designed on the basis of the differentially expressed and pathway gene sequences disclosed in Section 5.1 above, and/or on the basis cf the differentially expressed and pathway gene product sequences disclosed in this section.
  • Fusion proteins of the invention include, but are not limited to, IgFc fusion proteins which are useful, e.g., to stabilize the differentially expressed or pathway gene product such that the gene product has a prolonged half-life in vivo , as well as, e.g., fusions to any amino acid sequence that allows the fusion protein to be anchored to the cell membrane, fusions to an enzyme, fluorescent protein, luminescent protein, or a flag epitope protein or peptide which may be used to provide a marker function.
  • differentially expressed and pathway gene products of the present invention also include amino acid sequences encoded by the differentially expressed or pathway genes of the invention wherein domains encoded by one or more exons of the cDNA sequences of those genes, or fragments thereof, have been deleted.
  • the differentially expressed and pathway gene products of the invention can still further comprise post translational modifications, including, but not limited to glycosylations, acetylations, and myrisalations.
  • the differentially expressed or pathway gene products can be produced by synthetic techniques or via recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the differentially expressed or pathway gene polypeptides and peptides of the invention are described herein.
  • polypeptides and peptides of the invention can be synthesized or prepared by techniques well known in the art. See, for example, Creighton, 1983, "Proteins: Structures and Molecular Principles", W.H. Freeman and Co., N.Y., which is incorporated herein by reference in its entirety. Peptides can, for example, be synthesized on a solid support or in solution.
  • recombinant DNA methods which are well known to those skilled in the art can be used to construct expression vectors containing differentially expressed or pathway gene protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. which is incorporated by reference herein in their entirety, and Ausubel, 1989, supra.
  • RNA capable of encoding differentially expressed or pathway gene protein sequences can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.J. ed. , IRL Press, Oxford, which is incorporated by reference herein in its entirety.
  • a variety of host-expression vector systems can be utilized to express the differentially expressed or pathway gene coding sequences of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the differentially expressed or pathway gene protein of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g.. E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing differentially expressed or pathway gene protein coding sequences; yeast (e.g.
  • yeast expression vectors containing the differentially expressed or pathway gene protein coding sequences
  • insect cell systems infected with recombinant virus expression vectors (e.g. , baculovirus) containing the differentially expressed or pathway gene protein coding sequences
  • plant cell systems infected with recombinant virus expression vectors e.g. , cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g. , Ti plasmid
  • mammalian cell systems e.g.
  • COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g.. metallothionein promoter) or from mammalian viruses (e.g. , the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • promoters derived from the genome of mammalian cells
  • mammalian viruses e.g. , the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • a number of expression vectors can be advantageously selected depending upon the use intended for the differentially expressed or pathway gene protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2 : 1791), in which the differentially expressed or pathway gene protein coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.
  • pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) .
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione- agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the differentially expressed or pathway gene coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) .
  • Successful insertion of differentially expressed or pathway gene coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i.e. , virus lacking the proteinaceous coat coded for by the polyhedrin gene) .
  • non- occluded recombinant virus i.e. , virus lacking the proteinaceous coat coded for by the polyhedrin gene
  • These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g. , see Smith et al., 1983, J. Viol. 4j5:584; Smith, U.S. Patent No. 4,215,051).
  • a number of viral-based expression systems can be utilized.
  • the differentially expressed or pathway gene coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g. , the late promoter and tripartite leader sequence.
  • This chi eric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. , region El or E3) will result in a recombinant virus that is viable and capable of expressing differentially expressed or pathway gene protein in infected hosts, (e.g.
  • Specific initiation signals can also be required for efficient translation of inserted differentially expressed or pathway gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire differentially expressed or pathway gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals can be needed. However, in cases where only a portion of the differentially expressed or pathway gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided.
  • initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544) .
  • a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g.. glycosylation) and processing (e.g. , cleavage) of protein products can be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.
  • cell lines which stably express the differentially expressed or pathway gene protein can be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method can advantageously be used to engineer cell lines which express the differentially expressed or pathway gene protein.
  • Such engineered cell lines can be particularly useful in screening and evaluation of compounds 5 that affect the endogenous activity of the differentially expressed or pathway gene protein.
  • a number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine
  • any fusion protein may be readily
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus
  • the differentially expressed or pathway gene protein can be labeled, either directly or indirectly, to facilitate detection of a complex formed between the differentially expressed or pathway gene protein and a test substance.
  • suitable labeling systems can be used including but not limited to radioisotopes such as 125 I; enzyme labelling systems that generate a detectable colorimetric signal or light when exposed to substrate; and fluorescent labels.
  • Indirect labeling involves the use of a protein, such as a labeled antibody, which specifically binds to either a differentially expressed or pathway gene product.
  • a protein such as a labeled antibody
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by an Fab expression library.
  • fusion proteins can facilitate labeling (either direct or indirect) , immobilization, solubility and/or detection.
  • Fusion proteins which can facilitate solubility and/or expression, and can increase the blood half-life of the protein, can include, but are not limited to soluble Ig- tailed fusion proteins. Methods for engineering such soluble Ig-tailed fusion proteins are well known to those of skill in the art. See, for example U.S. Patent No. 5,116,964, which is incorporated herein by reference in its entirety. Further, in addition to the Ig-region encoded by the IgGl vector, the Fc portion of the Ig region utilized can be modified, by amino acid substitutions, to reduce complement activation and Fc binding. (See, e.g., European Patent No. 239400 Bl, August 3, 1994).
  • soluble Ig-tailed fusion proteins which can be produced are soluble Ig-tailed fusion proteins containing 103 gene products, 200 gene products or 10 gene products.
  • the 103 gene product or 200 gene contained within such fusion proteins can comprise, respectively, for example, the 103 gene extracellular or secreted domain or portions, preferably ligand-binding portions, thereof, or the 200 gene extracellular domain or portions, preferably ligand-binding 5 portions, thereof.
  • the 10 gene product contained within such fusion proteins can comprise, for example, one or more of the extracellular domains or portions, preferably ligand-binding portions, of the seven transmembrane domain sequence motif.
  • amino acid sequence of a soluble human 103 gene product is known.
  • amino acid residues which delineate the extracellular, transmembrane and cytoplasmic domains of the murine 103 gene products are also known. Still further, the amino acid sequences of murine and
  • 25 human 103 gene products are listed in SEQ ID NOS: 39 (murine full length, transmembrane 103 gene product) , 41 (murine extracellular domain of the full length, transmembrane product, plus amino terminal signal peptide) , 43 (murine intracellular domain of the full length, transmembrane
  • the murine 103 gene encodes two mRNA products, a 2.5 Kb
  • the human 103 gene encodes three mRNA products, an approximately 4.2 Kb transcript, an approximately 2.5 Kb transcript, and an approximately 1.4 Kb transcript (Kumar, S. 1997, Biochem Biophys Res Commun 235:474-478) .
  • Nucleotide sequences encoding 103 gene products are listed herein at SEQ ID NOS: 38 (nucleotide sequence encoding murine full length, transmembrane 103 gene product) , 40 (nucleotide sequence encoding extracellular domain of the murine 103 transmembrane gene product, plus amino terminal signal peptide) , 42 (nucleotide sequence encoding the intracellular domain of the murine 103 transmembrane gene product) , 46 (nucleotide sequence encoding the transmembrane domain of the murine 103 transmembrane gene product) , 49 (nucleotide sequence encoding the secreted murine 103 gene product) , and 44 (nucleotide sequence encoding human secreted/extracellular 103 gene product domain, plus amino terminal signal peptide) .
  • Murine and human 103 amino acid and nucleotide sequences are also depicted in FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, and FIG. 4H.
  • the nucleotide sequences of the 4.2 Kb and 2.5 Kb human 103 gene transcripts, as well as the amino acid sequences encoded by these transcripts, can be obtained utilizing standard techniques well known to those of skill in the art, including, e.g. , techniques such as those discussed herein. Therefore, by utilizing well known techniques, one of skill in the art would readily be capable of producing such soluble Ig-tailed 103 gene product fusion proteins.
  • the signal sequence, extracellular, transmembrane and cytoplasmic domains of both the murine and human 200 gene products have been elucidated and can be utilized in, for example, the construction of 200 gene product-Ig fusion proteins.
  • the 280 amino acid murine 200 gene product (FIG. 17; SEQ ID NO: 10) contains a signal sequence from approximately amino acid residue 1 to approximately amino acid residue 20, an extracellular domain from approximately amino acid residue 21 to approximately amino acid residue 192, a transmembrane domain from approximately amino acid residue 193 to amino acid residue 214, and a cytoplasmic domain from approximately amino acid residue 215 5 to amino acid residue 280.
  • the 301 amino acid human 200 gene product (FIG. 24; SEQ. ID.
  • NO: 24 contains a signal sequence from amino acid residue 1 to approximately 20, a mature extracellular domain from approximately amino acid residue 21 to 200, a transmembrane domain from approximately 0 amino acid residue 201-224 and a cytoplasmic domain from approximately amino acid residue 225 to 301. Given the elucidation of these domains, one of skill in the art would readily be capable of producing soluble Ig-tailed 200 gene product fusion proteins.
  • the Example presented, below, in 5 Section 10 describes the construction of murine and human 200 gene product-Ig fusion proteins.
  • the 338 amino acid residue 10 gene product (FIG.9, SEQ ID NO: 9) extracellular domains include 10 gene product amino acid residues from approximately amino acid residue 1 to 19, 0 approximately amino acid residue 74 to 87, approximately amino acid residue 153 to 187 and approximately amino acid residue 254 to 272.
  • 10 gene product domain information can be used, in conjunction with well-known techniques, such that one of skill in the art can readily be 5 capable of producing soluble Ig-tailed 10 gene fusion proteins comprising one or more 10 gene product extracellular domain regions and an Ig tail.
  • Described herein are methods for the production of antibodies capable of specifically recognizing one or more differentially expressed or pathway gene product epitopes.
  • Such antibodies can include, but are not limited to, _ polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies,
  • Fab fragments fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • the Ig tails of such antibodies can be modified to reduce complement activation and Fc binding. (See, for example, European Patent No. 239400 Bl, August 3, 1994).
  • Such antibodies can be used, for example, in the detection of a fingerprint, target, or pathway gene product in a biological sample, and can be used as part of diagnostic techniques. Alternatively, such antibodies can be utilized as part of an immune disorder treatment method, as described, below, in Section 5.9.
  • the antibodies can be used to modulate target gene activity, can be used to modulate TH cell subpopulation differentiation, maintenance and/or effector function, or, in the case of antibodies directed to cell surface epitopes, can be used to isolate a TH cell subpopulation of interest, for either depletion or augmentation purposes.
  • Such antibodies can also be utilized as part of a method for treatment of an ischemic disorder or injury, as described in Section 5.10.3.1, below.
  • the antibodies can be used to block or inhibit activity of one or more of the gene products of the invention, thereby reducing or inhibiting repair of certain ischemic tissues, for example carcinogenic tumors.
  • various host animals can be immunized by injection with a differentially expressed or pathway gene protein, or a portion thereof.
  • host animals can include but are not limited to rabbits, mice, and rats, to name but a few.
  • adjuvants can be used to increase the immunological response, depending on the host species, including but not limited to Freund ' s (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof.
  • an antigen such as target gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, can be immunized by injection with differentially expressed or pathway gene product supplemented with adjuvants as also described above.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al. , 1983, Immunology Today 4 . : 72; Cole et al. , 1983, Proc. Natl. Acad. Sci. USA 8Tj.2026-2030) , and the EBV- hybridoma technique (Cole et al.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454; U.S. Patent No. 4,816,567) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Antibody fragments which recognize specific epitopes can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries can be constructed (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Antibodies to the differentially expressed or pathway gene products can, in turn, be utilized to generate anti- idiotype antibodies that "mimic" such gene products, using techniques well known to those skilled in the art. (See, e.g. , Greenspan & Bona, 1993, FASEB J 7 (5) : 437-444; and Nissinoff, 1991, J. Immunol. 147 (8) : 2429-2438) .
  • receptor-type molecules e.g. , 10, 103 and 200 gene products
  • antibodies which bind to the ECD and competitively inhibit the binding of ligand to the receptor can be used to generate anti-idiotypes that "mimic" the ECD and, therefore, bind and neutralize the ligand.
  • Such neutralizing anti-idiotypes or Fab fragments of such anti- idiotypes can be used in therapeutic regimens of TH cell subpopulation-related disorders.
  • the animal-based model systems can be utilized to identify differentially expressed genes via the in vivo paradigm described, above, in Section 5.1.1.1.
  • Cell- and animal-based model systems can also be used to further characterize differentially expressed and pathway genes, as described, above, in Section 5.3. Such further characterization can, for example, indicate that a differentially expressed gene is a target gene.
  • Second, such assays can be utilized as part of screening strategies designed to identify compounds which are capable of ameliorating TH cell subpopulation-related disorder symptoms, as described, below.
  • the animal- and cell-based models can be used to identify drugs, pharmaceuticals, therapies and interventions which can be effective in treating immune disorders such as TH cell subpopulation-related disorders.
  • in Section 5.1.1.1 Cell- and animal-based model systems
  • Such assays can be
  • such animal models can be used to determine the LD 50 and the ED 50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential immune disorder treatments .
  • Animal-based model systems of TH cell subpopulation- related disorders can include both non-recombinant animals as well as recombinantly engineered transgenic animals.
  • Animal models for TH cell subpopulation-related disorders can include, for example, genetic models.
  • such animal models can include Leishmania resistance models, experimental allergic encephalomyelitis models and (BALB/c Cr x DBA/2Cr) FI mice. These latter mice develop a fatal disseminated disease by systemic infection with virulent Candida albicans associated with strong TH2-like 5 responses.
  • well known mouse models for asthma can be utilized to study the amelioration of symptoms caused by a TH2-like response.
  • murine acquired immunodeficiency syndrome (MAIDS; Kanagawa, B. et al., 1993, Science 262:240; Makino, M. et al., 1990, J. Imm. 144:4347) can be used for such studies.
  • mice see for example, Kaneshima, H. et al., 1994, Curr. Opin. Imm. 6:327-333
  • RAG-2-deficient blastocyst complementation technique Chen, J. et al., 1993, Proc. Natl. Acad. Sci. USA 90:4528-
  • mice containing, for example, humanized lymphocytes and/or which express target gene sequences can be utilized to produce mice containing, for example, humanized lymphocytes and/or which express target gene sequences. Still further, targeting techniques directed specifically to T cells, for example, the technique of Gu et al. (Gu, H. et)
  • THl or TH2 recipient mice results in TH effector cell migration to the airways and is associated with an intense neutrophilic (THl) and eosinophilic (TH2) lung mucosal inflammatory response.
  • THl intense neutrophilic
  • TH2 eosinophilic
  • Target gene sequences such as those described, above, in Section 5.4, in conjunction with techniques for producing transgenic animals that are well known to those of skill in the art.
  • target gene sequences can be introduced into, and overexpressed and/or misexpressed in, the genome of the animal of interest, or, if endogenous target gene sequences are present, they can either be overexpressed, misexpressed, or, alternatively, can be disrupted in order to underexpress or inactivate target gene expression.
  • the construction and characterization of 200 gene and 103 gene transgenic animals is described in Section 11, below.
  • the coding portion of the target gene sequence can be ligated to a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal and/or cell type of interest.
  • a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal and/or cell type of interest.
  • Such regulatory regions will be well known to those of skill in the art, and can be utilized in the absence of undue experimentation.
  • an endogenous target gene sequence For underexpression of an endogenous target gene sequence, such a sequence can be isolated and engineered such that when reintroduced into the genome of the animal of interest, the endogenous target gene alleles will be inactivated.
  • the engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target gene sequence into the animal's genome.
  • Gene targeting is discussed, below, in this Section. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g.
  • baboons, squirrels, monkeys, and chimpanzees can be used to generate animal models of TH cell subpopulation-related disorders.
  • Any technique known in the art can be used to introduce a target gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al. , 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al.
  • the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e. , mosaic animals.
  • the transgene can be integrated as a single transgene or in concatamers, e.g.. head-to-head tandems or head-to-tail tandems.
  • the transgene can also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci.
  • the transgene can also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following, for example, the teaching of Gu et al. (Gu, H. et al., 1994, Science 265:103-106) .
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • the level of mRNA expression of the transgene in the tissues of the transgenic animals can also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of target gene-expressing tissue, can also be evaluated immunocytochemically using antibodies specific for the target gene transgene gene product of interest.
  • target gene transgenic animals that express target gene mRNA or target gene transgene peptide (detected immunocytochemically, using antibodies directed against target gene product epitopes) at easily detectable levels can then be further evaluated to identify those animals which display characteristic TH cell subpopulation-related disorder-like symptoms, or exhibit characteristic TH cell subpopulation differentiation phenotypes.
  • THl-like-related disorder symptoms can include, for example, those associated with chronic inflammatory diseases and disorders, such as Crohn 's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease and sarcoidosis.
  • chronic inflammatory diseases and disorders such as Crohn 's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease and sarcoidosis.
  • TH2- like-related disorder symptoms can include, those associated with atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g. , leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.
  • atopic conditions such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g. , leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.
  • helminthic e.g. , leishmania
  • cellular phenotypes characteristic of TH cell subpopulation-related disorders can include, for example, differential cytokine expression characteristic of the TH cell subpopulation of interest. Further, such cellular phenotypes can include an assessment of a particular cell type's fingerprint pattern of expression and its comparison to known fingerprint expression profiles of the particular cell type in animals exhibiting specific TH cell subpopulation-related disorders. Such transgenic animals serve as suitable model systems for TH cell-related disorders.
  • target gene transgenic founder animals i.e. , those animals which express target gene proteins in cells or tissues of interest, and which, preferably, exhibit symptoms of TH cell subpopulation-related disorders
  • they can be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
  • breeding strategies include but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound target gene transgenics that express the target gene transgene of interest at higher levels because of the effects of additive expression of each target gene transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the possible need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the target gene transgene and the development of TH cell subpopulation- related disorder-like symptoms.
  • One such approach is to cross the target gene transgenic founder animals with a wild type strain to produce an FI generation that exhibits TH cell subpopulation-related disorder-like symptoms, such as those described above.
  • the FI generation can then be inbred in order to develop a homozygous line, if it is found that homozygous target gene transgenic animals are viable.
  • CELL-BASED ASSAYS Cells that contain and express target gene sequences which encode target gene protein, and, further, exhibit cellular phenotypes associated with a TH cell subpopulation- related disorder of interest, can be utilized to identify compounds that exhibit an ability to ameliorate TH cell subpopulation-related disorder symptoms.
  • Cellular phenotypes which can indicate an ability to ameliorate TH cell subpopulation-related disorder symptoms can include, for example, an inhibition or potentiation of cytokine or cell surface marker expression associated with the TH cell subpopulation of interest, or, alternatively, an inhibition or potentiation of specific TH cell subpopulations.
  • the fingerprint pattern of gene expression of cells of interest can be analyzed and compared to the normal, non-TH cell subpopulation-related disorder fingerprint pattern.
  • Those compounds which cause cells exhibiting TH cell subpopulation-related disorder-like cellular phenotypes to produce a fingerprint pattern more closely resembling a normal fingerprint pattern for the cell of interest can be considered candidates for further testing regarding an ability to ameliorate TH cell subpopulation-related disorder symptoms .
  • Cells which can be utilized for such assays can, for example, include non-recombinant cell lines, such as Dorris, AE7, D10.G4, DAX, Dl.l and CDC25 cell lines.
  • non-recombinant cell lines such as Dorris, AE7, D10.G4, DAX, Dl.l and CDC25 cell lines.
  • purified primary naive T cells derived from either transgenic or non-transgenic strains can also be used.
  • cells which can be used for such assays can also include recombinant, transgenic cell lines.
  • the TH cell subpopulation-related disorder animal models of the invention discussed, above, in Section 5.7.1, can be used to generate, for example, THl-like and/or TH2- like cell lines that can be used as cell culture models for the disorder of interest.
  • TH cell subpopulation-related disorder transgenic animals While primary cultures derived from TH cell subpopulation-related disorder transgenic animals can be utilized, the generation of continuous cell lines is preferred. For examples of techniques which can be used to derive a continuous cell line from the transgenic animals, see Small et al. , 1985, Mol. Cell Biol. 5:642-648.
  • cells of a cell type known to be involved in TH cell subpopulation-related disorders can be transfected with sequences capable of increasing or decreasing the amount of target gene expression within the cell.
  • target gene sequences can be introduced into, and overexpressed in, the genome of the cell of interest, or, if endogenous target gene sequences are present, they can either be overexpressed or, alternatively, can be disrupted in order to underexpress or inactivate target gene expression.
  • the coding portion of the target gene sequence can be ligated to a regulatory sequence which is capable of driving gene expression in the cell type of interest.
  • a regulatory sequence which is capable of driving gene expression in the cell type of interest.
  • an endogenous target gene sequence such a sequence can be isolated and engineered such that when reintroduced into the genome of the cell type of interest, the endogenous target gene alleles will be inactivated.
  • the engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target gene sequence into the cell's genome.
  • Gene targeting is discussed, above, in Section 5.7.1.
  • Transfection of target gene sequence nucleic acid can be accomplished by utilizing standard techniques. See, for example, Ausubel, 1989, supra. Transfected cells should be evaluated for the presence of the recombinant target gene sequences, for expression and accumulation of target gene mRNA, and for the presence of recombinant target gene protein production. In instances wherein a decrease in target gene expression is desired, standard techniques can be used to demonstrate whether a decrease in endogenous target gene expression and/or in target gene product production is achieved.
  • Cells to be utilized can, for example, be stimulated or activated as, described e.g. , in the Examples presented below.
  • the following assays are designed to identify compounds that bind to target gene products, bind to other cellular proteins that interact with a target gene product, and to compounds that interfere with the interaction of the target gene product with other cellular proteins.
  • a target gene product which are or are predicted to be transmembrane receptor-type proteins
  • such techniques can identify ligands for such receptors.
  • a compound which binds a 103 gene product (a 103 gene product ligand, for example) can act as the basis for amelioration of such TH2-like-specific disorders as asthma or allergy, given that gene 103 expression is TH2-specific.
  • a 200 gene product ligand can, for example, act as the basis for amelioration of
  • a 10 gene product ligand can, for example, act as the basis for amelioratoin of a wide range of T cell disorders, given the TH inducible nature of it gene expression pattern. Any such binding compound can act as a marker for the presence of TH cell subpopulations.
  • a compound which binds the 103 gene product can act as a marker, for example, a diagnostic marker, for TH2 cells, e.g. , for TH2 cell differentiation.
  • Compounds can include, but are not limited to, other cellular proteins. Further, such compounds can include, but 5 are not limited to, peptides such as, for example, soluble peptides, including, but not limited to, Ig-tailed fusion peptides, comprising extracellular portions of target gene product transmembrane receptors, and members of random peptide libraries (see, e.g. , Lam, K.S. et al. , 1991, Nature
  • receptor-type including, but not limited to polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof) , and small organic or inorganic molecules.
  • receptor-type including, but not limited to polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof
  • small organic or inorganic molecules include, but not limited to polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof.
  • such compounds can include organic molecules (e.g. , peptidomimetics) that bind to the ECD and either mimic the activity triggered by the natural ligand (i.e. , agonists) ; as well as peptides, antibodies or fragments thereof, and other organic compounds that mimic the natural ligand (i.e. , agonists) ; as well as peptides, antibodies or fragments thereof, and other organic compounds that mimic the
  • Computer modelling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate target or pathway
  • such active sites might typically be ligand binding sites
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the co plexed ligand is found.
  • the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.
  • the methods of computer based numerical modelling can be used to complete the structure or improve its accuracy.
  • Any recognized modelling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a seach can be manual, but is preferably computer assisted. These compounds found from this search are potential target or pathway gene product modulating compounds .
  • these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modelling methods described above applied to the new composition.
  • the altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results.
  • systematic variations in composition such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • Further experimental and computer modeling methods useful to identify modulating compounds based upon identification of the active sites of target or pathway gene or gene products and related transduction and transcription factors will be apparent to those of skill in the art.
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • Soc. 111:1082-1090 Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, CA. ) , Allelix, Inc. (Mississauga, Ontario, Canada) , and Hypercube, Inc. (Cambridge, Ontario) . Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA, once that region is identified.
  • Such compounds would bring about an effective increase in the level of target gene activity, thus ameliorating symptoms.
  • compounds that bind target gene protein can be identified that inhibit the activity of the bound target gene protein.
  • Assays for identifying additional compounds as well as for testing the 5 effectiveness of compounds identified by, for example, techniques, such as those described in Section 5.8.1-5.8.3, are discussed, below, in Section 5.8.4.
  • In vitro systems can be designed to identify compounds capable of binding the target gene products of the invention.
  • Compounds identified can be useful, for example, in modulating the activity of wild type and/or mutant target _ gene products, can be useful in elaborating the biological function of target gene products, can be utilized in screens for identifying compounds that disrupt normal target gene product interactions, or can in themselves disrupt such interactions.
  • the principle of the assays used to identify compounds that bind to the target gene product involves preparing a reaction mixture of the target gene product and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a
  • the target gene product can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly.
  • microtiter plates can conveniently be utilized as the solid phase.
  • the anchored component can be immobilized by non-covalent or covalent attachments.
  • Non- covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface.
  • the surfaces can be prepared in advance and stored.
  • the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g. , by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g. , using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody) .
  • a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g. , using an immobilized antibody specific for target gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
  • an immobilized antibody specific for target gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
  • techniques such as those described in this section can be utilized to identify compounds which bind to the 103 gene product. For example, a 103 gene product can be contacted with a compound for a time sufficient to form a 103 gene product/compound complex and then such a complex can be detected.
  • the compound can be contacted with the 103 gene product in a reaction mixture for a time sufficient to form a 103 gene product/compound complex, and then such a complex can be separated from the reaction mixture.
  • 103 gene products which can be utilized for such methods are, for example, rat, murine and human 103 gene products, including, but not limited to the 103 gene products listed in SEQ ID NOS: 39, 41, 43, 45, 47 or 48 (with or without signal peptide sequences) or a naturally occurring variant thereof.
  • naturally occurring variant refers to an amino acid sequence homologous to the 103 gene product in the same or a different species, such as, for example, an allelic variant of the 103 gene product which maps to the same chromosomal location as the nucleotide sequences encoding the 103 gene products of SEQ ID NOS: 39, 41, 43, 45, 47 or 48, or a location syntenic to such a location.
  • allelic variants which can be utilized herein are allelic variant sequences encoded by a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence encoding the 103 gene products described above (that is SEQ ID NOS: 39, 41, 43, 45, 47 or 48, such as, for example, SEQ ID NOS: 38, 40, 42, 44,
  • Any method suitable for detecting protein-protein interactions can be employed for identifying novel target protein-cellular or extracellular protein interactions.
  • the target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins.
  • macromolecules can include, but are not limited to, nucleic acid molecules and those proteins identified via methods such as those described, above, in Section 5.8.2.
  • binding partners such cellular and extracellular macromolecules are referred to herein as "binding partners”.
  • binding partners Compounds that disrupt such interactions can be useful in regulating the activity of the target gene protein, especially mutant target gene proteins.
  • Such compounds can include, but are not limited to molecules such as antibodies, peptides, and the like, as described, for example, in Section 5.8.1. above.
  • the basic principle of the assay systems used to identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the target gene product and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex.
  • the reaction mixture is prepared in the presence and absence of the test compound.
  • the test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of target gene product and its cellular or extracellular binding partner.
  • Control reaction mixtures are incubated without the test compound or with a placebo.
  • the formation of any complexes between the target gene protein and the cellular or extracellular binding partner is then detected.
  • the formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene protein and the interactive binding partner.
  • complex formation within reaction mixtures containing the test compound and normal target gene protein can also be compared to complex formation within reaction mixtures containing the test compound and a mutant target gene protein. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene proteins.
  • the assay for compounds that interfere with the interaction of the target gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g. , by competition, can be identified by conducting the reaction in the presence of the test substance; i.e. , by adding the test substance to the reaction mixture prior to or simultaneously with the target gene protein and interactive cellular or extracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.
  • either the target gene protein or the interactive cellular or extracellular binding partner is anchored onto a solid surface, while the non- anchored species is labeled, either directly or indirectly.
  • the anchored species can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished simply by coating the solid surface with a solution of the target gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored.
  • the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g. , by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre- labeled, an indirect label can be used to detect complexes anchored on the surface; e.g.
  • the antibody in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody.
  • test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g. , using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds which inhibit complex or which disrupt preformed complexes can be identified.
  • a homogeneous assay can be used.
  • a preformed complex of the target gene protein and the interactive cellular or extracellular binding partner is prepared in which either the target gene product or its binding partner is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g. , U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays) .
  • the addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt target gene protein/cellular or extracellular binding partner interaction can be identified.
  • the target gene product can be prepared for immobilization using recombinant DNA techniques described in Section 5.5, above.
  • the target gene coding region can be fused to a glutathione-S- transferase (GST) gene using a fusion vector, such as pGEX- 5X-1, in such a manner that its binding activity is maintained in the resulting fusion protein.
  • GST glutathione-S- transferase
  • the interactive cellular or extracellular binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above, in Section 5.6.
  • This antibody can be labeled with the radioactive isotope 125 I, for example, by methods routinely practiced in the art.
  • a heterogeneous assay e.g.
  • the GST-target gene fusion protein can be anchored to glutathione-agarose beads.
  • the interactive cellular or extracellular binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
  • unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
  • the interaction between the target gene protein and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
  • the GST-target gene fusion protein and the interactive cellular or extracellular binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
  • the test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the target gene product/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
  • these same techniques can be employed using peptide fragments that correspond to the binding domains of the target gene product and/or the interactive cellular or extracellular binding partner (in cases where the binding partner is a protein) , in place of one or both of the full length proteins.
  • Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding.
  • one protein can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain can remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the cellular or extracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.
  • a proteolytic enzyme such as trypsin
  • a target gene product can be anchored to a solid material as described, above, in this Section, by making a GST-target gene fusion protein and allowing it to bind to glutathione agarose beads.
  • the interactive cellular or extracellular binding partner can be labeled with a radioactive isotope, such as 35 S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-target gene 5 fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the cellular or extracellular binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well known methods. Peptides so identified can be produced 0 synthetically or fused to appropriate facilitative proteins using well known recombinant DNA technology.
  • Any of the binding compounds including but not limited to, compounds such as those identified in the foregoing assay systems, can be tested for the ability to ameliorate symptoms of immune disorders e.g. , TH cell subpopulation-related disorders.
  • Cell-based and animal model-based assays for the 0 identification of compounds exhibiting such an ability to ameliorate immune disorder symptoms are described below.
  • cell-based assays for the identification of compounds which modulate target gene product function in instances where the target gene product is a receptor having 5 a seven transmembrane domain sequence, such as, for example, that of the 10 gene product, are described, below, in Section 5.8.4.1.
  • cell-based systems such as those described, above, in Section 5.7.2, can be used to identify compounds 0 which can act to ameliorate TH cell subpopulation-related disorder symptoms.
  • such cell systems can be exposed to a compound, suspected of exhibiting an ability to ameliorate the disorder symptoms, at a sufficient concentration and for a time sufficient to elicit such an _ amelioration in the exposed cells.
  • the cells are examined to determine whether one or more of the TH cell subpopulation-related disorder-like cellular phenotypes has been altered to resemble a phenotype more likely to produce a lower incidence or severity of disorder symptoms. Additional cell-based assays are discussed, below, in Section 5.8.4.1.
  • any TH2 or TH2-like cell system can be utilized.
  • compounds can be assayed for their ability to modulate the TH2-like phenotype of such cells, such that the cells exhibit loss of a TH2-like phenotype.
  • Compounds with such TH2 modulatory capability represent ones which can potentially exhibit the ability to ameliorate asthma-related symptoms in vivo.
  • Example presented in Section 12, below, describes the successful utilization of a 103 gene product/Ig fusion protein, as well as the successful use of a monoclonal antibody directed against the extracellular domain of the 103 gene product to ameliorate symptoms of asthma in an accepted animal model of asthma .
  • animal-based systems such as those described, above, in Section 5.7.1, can be used to identify compounds capable of ameliorating TH cell subpopulation- related disorder-like symptoms.
  • Such animal models can be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions which can be effective in treating such disorders.
  • animal models can be exposed to a compound, suspected of exhibiting an ability to ameliorate TH cell subpopulation-related disorder symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of the symptoms in the exposed animals.
  • the response of the animals to the exposure, and thus the efficacy of the compound in question can be monitored by assessing the reversal of disorders associated with TH cell subpopulation-related disorders of interest.
  • any treatments which reverse any aspect of TH cell subpopulation- related disorder-like symptoms should be considered as candidates for corresponding human TH cell subpopulation- related disorder therapeutic intervention.
  • Dosages of test agents can be determined by deriving dose-response curves, as discussed in Section 5.11, below.
  • Gene expression patterns can be utilized in conjunction with either cell-based or animal-based systems, to assess the ability of a compound to ameliorate TH cell subpopulation- related disorder-like symptoms.
  • the expression pattern of one or more fingerprint genes can form part of a fingerprint profile which can be then be used in such an assessment.
  • Fingerprint profiles are described, below, in Section 5.12. Fingerprint profiles can be characterized for known states, either TH cell subpopulation-related disorder states, or normal TH cell differentiative states, within the cell- and/or animal-based model systems.
  • the 10 gene product (FIG. 9; SEQ ID NO: 9) is an example of a seven transmembrane domain target gene product.
  • the 10 gene product will be used to illustrate the methods described in this Section.
  • the compounds tested may be, for example, compounds such as those identified via the assays described, above, in Sections 5.8.1 to 5.8.3.
  • Such compounds may include, but are not limited to peptides such as, for example, soluble 0 peptides, including, but not limited to, Ig-tailed fusion peptides, comprising extracellular portions of target gene product transmembrane receptors, and members of random peptide libraries (see, e.g. , Lam, K.S. et al. , 1991, Nature 354:82-84 ; Houghten, R. et al., 1991, Nature 354:84-86) made _ of D-and/or L-configuration amino acids, phosphopeptides
  • antibodies including, but not limited to polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof) , and small organic or inorganic molecules.
  • the assays described herein are functional assays which identify compounds that affect the receptor target gene's activity by affecting the level of intracellular calcium release within cells expressing such seven transmembrane domain receptor target protein (e.g. , the 10 gene product) . Intracellular calcium release is measured because such seven transmembrane domain receptors tend to be G protein-coupled receptors and because activation of these receptors leads to a G protein-mediated intracellular calcium release.
  • Modulation i.e. , agonization or antagonization
  • agonization or antagonization of the receptor target gene product function
  • the assays comprise contacting a seven transmembrane domain receptor target gene-expressing cell with a test compound and measuring the level of intracellular calcium.
  • Those compounds which produce an intracellular calcium profile which differs from that which the cell would exhibit in the absence of the compound represent either agonists or antagonists.
  • An agonist compound would cause an increase in intracellular calcium levels relative to control cells while an antagonist would result in a decrease in intracellular calcium levels relative to control cells.
  • any cell expressing a seven transmembrane receptor target gene product may be used herein, it is preferred that cells be used whose intracellular calcium levels may readily measured.
  • Xenopus oocytes due to their large size, are among such preferred cells because they can easily be injected with intracellular calcium reporter compounds.
  • myeloma cells may be utilized.
  • reporter compounds include, but are not limited to, calcium-binding agents such as the well known FURA-2 and INDO-2.
  • FURA- 2/calcium complexes and INDO-2/calcium complexes fluoresce, making possible the measurement of differences in intracellular calcium levels.
  • the Xenopus oocytes should be transfected with nucleotide sequences encoding the target protein of interest (e.g. , the 10 gene product) .
  • the cells can be transfected and express the sequence of interest via techniques which are well known to those of s ⁇ ill in the art and which may include, for example, techniques such as those described, above, in Section 5.5.
  • Xenopus oocytes can be injected with RNA encoding the target gene product of interest such that the injected oocytes will express the gene product.
  • the assays described in this Section may, first, be used to identify compounds which act as agonists of the target gene product of interest, e.g. , the 10 gene product.
  • “Agonist”, as used herein, refers to a compound which modulates target gene product activity by increasing the target gene product's activity, as evaluated by the compound's ability to bring about an increase in calcium influx, leading to an increase intracellular calcium levels.
  • agonists can be, for example, the natural ligand for the receptor target gene product, e.g.. the natural ligand for the 10 gene product.
  • Agonists identified via such assays may act as useful therapeutic agents for the amelioration of a wide range of T cell-related disorders, including, for example, TH cell subpopulation-related disorders, in instances whereby such disorders are caused by a reduced or absent level of target gene product activity.
  • Any of the agonist compounds identified herein can be used, for example, as part of the treatment methods described in Section 5.9.2, below. Further, such agonists can be used to identify antagonists of the receptor target gene product of interest, e.g. , as described, below.
  • Antagonist refers to a compound which modulates target gene product activity by decreasing the target gene product's activity, as evaluated by the compound's ability to bring about a decrease in calcium influx. Antagonists identified via such assays may act as useful therapeutic agents for the amelioration of a wide range of T cell-related disorders, including, for example, TH cell subpopulation-related disorders, in instances whereby the disorder is caused by an increased or inappropriate level of target gene product activity.
  • An antagonist screen may be performed utilizing target gene product-expressing cells as described, above, and which include, but are not limited to, such cells as 10 gene- expressing cells, for example, 10 gene-expressing Xenopus oocytes.
  • the cells utilized in the antagonist assay can be cells which express the mutant receptor target gene product involved in causing the T cell-related disorder.
  • a target gene- expressing cell is contacted with 1) an agonist of the target gene product and 2) a test compound for a given period of time.
  • the level of intracellular calcium is then measured in the cells and in cells which have been contacted with agonist alone.
  • a test compound is considered to be an antagonist if the level of intracellular calcium release in the presence of the test compound is lower than the level of intracellular calcium release in the absence of the test compound.
  • any of the antagonist compounds identified herein can be used, for example, as part of the treatment methods described, below, in Section 5.9.1.
  • the potential antagonist compounds of the seven transmembrane domain receptor target gene products described herein are peptides which contain one or more of the receptor target gene product's extracellular domains, preferably those domains are domains which are responsible for ligand-binding such that the peptides act to compete with the endogenous receptor for ligand.
  • extracellular domains include from approximately 10 gene product amino acid residue 1 to 19, amino acid residue 74 to 87, amino acid residue 153-187 and amino acid residue 254 to 272.
  • extracellular domain antagonist compounds may comprise soluble Ig-tailed fusion 5 proteins which may be produced by utilizing techniques such as those described, above, in Section 5.5. Additionally, antibodies directed against the extracellular portion of the 10 gene product may reduce 10 gene product function by, for example, blocking ligand binding.
  • compositions which can be used to ameliorate immune disorder symptoms via, for
  • a modulation of the TH cell subpopulation of interest can be of a positive or negative nature, depending on the specific situation involved, but each modulatory event yields a net result in which symptoms of the immune disorder are ameliorated.
  • described 2 _ ⁇ 0 below are methods for the modulation of TH cell responsiveness to antigen.
  • “Negative modulation”, as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene
  • the term, as used herein, refers to a depletion of the T cell subpopulation (e.g. , via a reduction in the number of cells belonging to the TH cell subpopulation) relative to the number present in the absence
  • “Positive modulation”, as used herein, refers to an increase in the level and/or activity of target gene product relative to the level and/or activity of the gene product in
  • the term, as used herein, refers to a stimulation of the T cell subpopulation (e.g. , via an increase in the number of cells belonging to the TH cell subpopulation) , relative to the number present in the absence of the modulatory treatment.
  • stimulation is as defined, above, in Section 3. It is possible that a TH cell subpopulation-related disorder or other immune disorder, can occur as a result of normal target gene activity during the course of, for example, exposure to a certain antigen which elicits an immune response that leads to the development of the disorder. For example, the TH2-like-related disorders, asthma and allergy, are likely candidates of disorders having such a mechanism.
  • a disorder can be brought about, at least in part, by an abnormally high level of target gene product, or by the presence of a target gene product exhibiting an abnormal activity.
  • a technique which elicits a negative modulatory effect i.e. , brings about a reduction in the level and/or activity of target gene product, or alternatively, brings about a depletion of the TH cell subpopulation (e.g. , via a physical reduction in the number of cells belonging to the TH cell subpopulation) , would effect an amelioration of TH cell subpopulation-related disorder symptoms in either of the above scenarios.
  • Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels, (either normal or abnormal) , and for the reduction in the number of specific TH cell subpopulation cells are discussed in Section 5.9.1, below.
  • a TH cell subpopulation-related disorder or other immune disorders can be brought about, at least in part, by the absence or reduction of the level of target gene expression, a reduction in the level of a target gene product's activity, or a reduction in the overall number of cells belonging to a specific TH cell subpopulation.
  • a technique which elicits a positive modulatory effect i.e. , brings about an increase in the level of target gene expression and/or the
  • a stimulation of the TH cell subpopulation e.g. , via a physical increase in the number of cells belonging to a TH cell subpopulation
  • a reduction in the overall number of THl- like cells relative to TH2-like cells within a HIV-infected individual can correlate with the progression to AIDS (Clerci, M. et al., 1993, J. Clin. Invest. 91:759; Clerci, M. et al., 1993, Science 262:1721; Maggi, E. et al., 1994, Science 265: 244) .
  • a treatment capable of increasing the number of THl-like cells relative to TH2-like cells within an HIV-infected individual may, therefore, serve to prevent or slow the progression to disease.
  • THl or THl-like related immune disorders include chronic inflammatory diseases and disorders, such as Crohn ' s disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease and sarcoidosis.
  • TH2 or TH2-like related disorders include atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g. , leishmaniasis) and certain viral infections, including HIV, and bacterial infections, including tuberculosis and lepromatous leprosy.
  • helminthic e.g. , leishmaniasis
  • viral infections including HIV
  • bacterial infections including tuberculosis and lepromatous leprosy.
  • the methods described herein can additionally be utilized the modulate the level of responsiveness, for example, responsiveness to antigen, of a TH cell subpopulation. Such methods are important in that many immune disorders involve inappropriate rather than insufficient immune responses.
  • disorders such as atopic, IgE-mediated allergic conditions, including asthma, pathogen susceptibilities and chronic inflammatory disease, involve strong but counterproductive TH2-mediated immune responses.
  • inappropriate THl-mediated immune responses to self-antigens is central to the development of such disorders as multiple sclerosis, psoriasis, insulin dependent diabetes, Hashimoto's thyroiditis and Crohn ' s disease.
  • Methods for modulating TH cell responsiveness can comprise, for example, contacting a compound to a TH cell so that the responsiveness of the T helper cell is modulated relative to the responsiveness of the T helper cell in the absence of the compound.
  • the modulation can increase or decrease the responsiveness of the TH cell. Any of the techniques described, below, in Sections 5.9.1-5.9.3.2 can be utilized to effect an appropriate modulation of TH cell responsiveness .
  • the methods and techniques described herein can also be used for treating, ameliorating, or modulating symptoms associated with mast cell-related processes or disorders or certain ischemia disorders or injuries.
  • techniques which increase the expression or activity of certain gene products of the invention whose activity is involved in the repair of ischemic injury or damage can be used to treat tissue or organ damage produced by an ischemic disorder or injury.
  • techniques or methods which inhibit the expression or activity of target gene products of the invention can be used to block or inhibit the repair of ischemic tissue or organs.
  • Such techniques are useful, for example, to treat ischemic or infarcated tissue, such as a cancerous tumor, to increase damage or injury to such tissue, e.g., during treatment such as chemotherapy.
  • ischemic disorders or injuries whose symptoms can be ameliorated are ischemic renal disease and myocardial ischemia, such as angina pectoris, as well as ischemic injuries to other tissues including, but by no means limited to, the brain (as in a stroke) , spleen, intestine, lung, and testes.
  • ischemic renal disease and myocardial ischemia such as angina pectoris
  • ischemic injuries to other tissues including, but by no means limited to, the brain (as in a stroke) , spleen, intestine, lung, and testes.
  • Such techniques can also be used to treat, or to enhance ischemic injuries to tumors, including tumors of the ovary or uterus.
  • the methods described herein can additionally be used to treat or prevent ischemic damage or injury to transplanted organs, such as transplanted kidneys, lungs, hearts, livers, and pancrease, or grafts, such as skin grafts.
  • transplanted organs such as transplanted kidneys, lungs, hearts, livers, and pancrease
  • grafts such as skin grafts.
  • Section 13 herein, to play a critical role in the resolution (i.e., the repair) of injury following ischemia reperfusion.
  • the 200 gene and its products can, therefore, be utilized in the treatment of ischemic disorders and injuries.
  • a gene 200 product, or functional portions thereof can be utilized either directly or indirectly to stimulate or increase the repair of injury to tissue or organs resulting from an ischemic injury or disorder.
  • successful treatment of certain immune disorders can be brought about by techniques which serve to inhibit the expression or activity of target gene products, or which, alternatively, serve to reduce the overall number of cells belonging to a specific TH cell subpopulation.
  • techniques which serve to inhibit the expression or activity of target gene products of the present invention, including the 200 gene product can be used to inhibit the repair or recovery of certain ischemic tissue.
  • compounds such as those identified through assays described, above, in Section 5.8, which exhibit negative modulatory activity can be used in accordance with the invention to ameliorate certain TH cell subpopulation- related disorder symptoms, or, alternatively, to inhibit the repair or recovery of ischemic tissue.
  • compounds such as those identified through assays described, above, in Section 5.8, which exhibit negative modulatory activity, can be used in accordance with the invention to ameliorate certain TH cell subpopulation- related disorder symptoms, or, alternatively, to inhibit the repair or recovery of ischemic tissue.
  • such molecules can include, but are not limited to peptides (such as, for example, peptides representing soluble extracellular portions of target gene product transmembrane receptors) , phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof) .
  • antibodies directed against a 103 gene product preferably an extracellular or extracellular portion of a 103 gene product, can be utilized. Techniques for the determination of effective doses and administration of such compounds are described, below, in Section 5.11.
  • antisense and ribozyme molecules which inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity.
  • triple helix molecules can be utilized in reducing the level of target gene activity.
  • Such techniques can take advantage of, for example, novel cell surface markers which are specific to the TH cell subpopulation to be depleted, and can include in vivo or in vitro targeted destruction, or, alternatively, selective purification away, of the TH cell subpopulation of interest.
  • TH cell subpopulation-related sequences identified by the methods described by the present invention is a gene designated herein as the 103 gene, as discussed in the Example presented in Section 7, below.
  • the 103 gene is demonstrated herein to represent a TH2-specific gene in that 103 gene expression is found to be absent THl cells as well as all other tissues tested. Further, at least one of the proteins produced by the 103 gene is a transmembrane protein. The 103 gene and its products can, therefore, be utilized in the treatment of TH2 cell subpopulation-related disorders.
  • a 103 gene product or portions thereof can be utilized, either directly or indirectly, to ameliorate conditions involving inappropriate IgE immune responses, including, but not limited to the symptoms which accompany atopic conditions such as allergy and/or asthma.
  • IgE-type antibodies are produced by stimulated B cells which require, at least in part, IL-4 produced by the TH2 cell subpopulation. Therefore, any treatment, including, for example, the use of a gene 103 product or portion thereof, which reduces the effective concentration of secreted IL-4, e.g. , by reducing the number or activity of TH2 cells, can bring about a reduction in the level of circulating IgE, leading, in turn, to the amelioration of the conditions stemming from an inappropriate IgE immune response.
  • TH2 specific 103 gene products can be used to effect such a reduction in the activity and/or effective concentration of TH2 cells.
  • natural ligands, derivatives of natural ligands and antibodies which bind to the 103 gene product can be utilized to reduce the number of TH2 cells present by either physically separating such cells away from other cells in a population, thereby deleting the TH2 cell subpopulation, or, alternatively, by targeting the specific destruction of TH2 cells.
  • Such techniques are discussed, below, in Section 5.9.3. Further, such compounds can be used to inhibit the proliferation of TH2 cells.
  • compounds such as 103 gene sequences or gene products can be utilized to reduce the level of TH2 cell activity, cause a reduction in IL-4 production, and, ultimately, bring about the amelioration of IgE related disorders.
  • compounds can be administered which compete with endogenous ligand for the 103 gene product.
  • the resulting reduction in the amount of ligand-bound 103 gene transmembrane protein will modulate TH2 cellular activity.
  • Compounds which can be particularly useful for this purpose include, for example, soluble proteins or peptides, such as peptides comprising the extracellular domain, or portions and/or analogs thereof, of the gene 103 product, including, for example, soluble fusion proteins such as Ig-tailed fusion proteins. (For a discussion of the production of Ig-tailed fusion proteins see, for example, U.S. Patent No. 5, 116,964.) Production of a 103 gene product/Ig fusion is described in Section 10, below. Further, use of a 103 gene product/Ig fusion to successfully ameliorate symptoms in an accepted animal model for asthma is described in Section 12, below.
  • the novel 200 gene which encodes a receptor target gene product that is a member of the Ig superfamily, exhibits a THl-specific pattern of gene expression.
  • the 200 gene especially the human 200 gene, and its products can, therefore, be utilized in the treatment of THl cell subpopulation-related disorders such as, for example, chronic inflammatory diseases, psoriasis, graft rejection and graft versus host disease.
  • the treatment of such disorder may require a reduction in the activity and/or effective concentration of the THl cell subpopulation involved in the disorder of interest.
  • natural ligands, derivatives of natural ligands and antibodies which bind to the 200 gene product can be utilized to reduce the number of THl cells present by either physically separating such cells away from other cells in a population, thereby deleting the THl cell subpopulation, or, alternatively, by targeting the specific destruction of THl cells.
  • Such techniques are discussed, below, in Section 5.10.3. Further, such compounds can be used to inhibit the proliferation of THl cells.
  • compounds can be administered which compete with endogenous ligand for the 200 gene product. Such compounds would bind to and "neutralize" circulating ligand. The resulting reduction in the amount of ligand- bound 200 gene transmembrane protein will modulate THl cellular activity. Further, reduction in the amount of ligand bound 200 gene transmembrane protein will also inhibit the resolution and repair of ischemic tissue.
  • soluble proteins or peptides such as peptides comprising the extracellular domain, or portions and/or analogs thereof, of the gene 200 product, including, for example, soluble fusion proteins such as Ig- tailed fusion proteins or antibodies.
  • soluble fusion proteins such as Ig- tailed fusion proteins or antibodies.
  • peptides corresponding to the ECD of the 200 gene product, soluble deletion mutants of 200 gene product, or either of these 200 gene product domains or mutants fused to another polypeptide can be utilized.
  • another polypeptide e.g. , an IgFc polypeptide
  • anti-idiotypic antibodies or Fab fragments of antiidiotypic antibodies that mimic the 200 gene product ECD and neutralize 200 gene product ligand can be used.
  • Such 200 gene product peptides, proteins, fusion proteins, anti-idiotypic antibodies or Fabs are administered tc a subject in amounts sufficient to neutralize the gene product and thereby effectuate an amelioration of a T cell subpopulation-related disorder, or an inhibition of repair of ischemic tissues.
  • the Fc region of the Ig portion of the fusion protein may be further modified to reduce immunoglobulin effector function.
  • nucleotide sequences encoding the fusion protein may be modified to encode fusion proteins which replace cysteine residues in the hinge region with serine residues and/or amino acids within the CH2 domain believed to be required for IgC binding to FC receptors and complement activation.
  • cells that are genetically engineered to express such soluble or secreted forms of 200 gene product may be administered to a patient, whereupon they will serve as "bioreactors" in vivo to provide a continuous supply of the 200 gene product ligand neutralizing protein.
  • Such cells may be obtained from the patient or an MHC compatible donor and can include, but are not limited to fibroblasts, blood cells (e.g. , lymphocytes) , adipocytes, muscle cells, endothelial cells etc.
  • the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence for the 200 gene product peptide, or 2C0 gene product fusion proteins
  • the 200 gene product coding sequence can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression and secretion of the 200 gene peptide or fusion protein.
  • the engineered cells which express and secrete the desired 200 gene product can be introduced into the patient systemically, e.g. , in the circulation, or intrapertioneally .
  • the cells can be incorporated into a matrix and implanted in the body, e.g. , genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a vascular graft.
  • genetically engineered fibroblasts can be implanted as part of a skin graft; and genetically engineered endothelial cells can be implanted as part of a vascular graft.
  • the cells to be administered are non-autologous cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • the 10 gene product is identified herein as a receptor target gene product having a seven transmembrane domain sequence motif. Further, the 10 gene is shown to exhibit a TH inducible pattern of expression, meaning that 10 gene expression increases in both THl and TH2 cell subpopulations in response to stimulation and can important to T cell responses in general.
  • the 10 gene and its products can, therefore, be utilized in the treatment of a wide T cell- related disorders. Techniques such as those described, above, for the 103 and the 200 genes and gene products can also be utilized for the amelioration of disorders in which 10 gene expression is involved.
  • antisense ribozyme
  • triple helix molecules Such molecules can be designed to reduce or inhibit either wild type, or if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to target or pathway gene mRNA.
  • the antisense oligonucleotides will bind to the complementary target or pathway gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be) . One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Oligonucleotides that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
  • sequences complementary to the 3 ' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R. , 1994, Nature 372:333-335.
  • oligonucleotides complementary to either the 5'- or 3'- non-translated, non-coding regions of target or pathway genes could be used in an antisense approach to inhibit translation of endogenous target or pathway gene mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least
  • nucleotides 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single- stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g. , for targeting host cell receptors in vivo) , or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethy1-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2 , 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyaminomethyl-2-thiouracil , beta-D-mannos
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an -anomeric oligonucleotide.
  • An -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2'- O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett.
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
  • the antisense molecules should be delivered to cells which express the target or pathway gene in vivo .
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g. , antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g. , antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically .
  • a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.
  • the use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target or pathway gene transcripts and thereby prevent translation of the target or pathway gene mRNA.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive.
  • Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441- 1445) , the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.
  • Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site. Alternatively, viral vectors can be used which selectively infect the desired tissue.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (For a review see, for example Rossi, J. , 1994, Current Biology 4 . :469-471).
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
  • the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety.
  • Ribozyme molecules designed to catalytically cleave target or pathway gene mRNA transcripts can also be used to prevent translation of target or pathway gene mRNA and expression of target or pathway gene.
  • Ribozyme molecules designed to catalytically cleave target or pathway gene mRNA transcripts can also be used to prevent translation of target or pathway gene mRNA and expression of target or pathway gene.
  • PCT International Publication WO90/11364 published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225.
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target or pathway gene mRNAs, the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
  • the sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target or pathway gene mRNA; i.e.. to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as 5 the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-
  • Cech-type ribozymes such as 5 the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986
  • Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in target or pathway gene.
  • the ribozymes can be composed of modified oligonucleotides (e.g. for improved oligonucleotides).
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected
  • 25 cells will produce sufficient quantities of the ribozyme to destroy endogenous target or pathway gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • the technique can also efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense,
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy methods such as those described, below, in Section 5.9.2 that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized.
  • the target gene encodes an extracellular protein
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2 ' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • Endogenous target and/or pathway gene expression can also be reduced by inactivating or "knocking out" the target and/or pathway gene or its promoter using targeted homologous recombination.
  • endogenous target and/or pathway gene expression can also be reduced by inactivating or "knocking out" the target and/or pathway gene or its promoter using targeted homologous recombination.
  • a mutant, non-functional target and/or pathway gene flanked by DNA homologous to the endogenous target and/or pathway gene (either the coding regions or regulatory regions of the target and/or pathway gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express target and/or pathway gene in vivo . Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target and/or pathway gene.
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target and/or pathway gene (e.g.
  • T cell subpopulation-related disorder animal models See Thomas & Capecchi 1987 and Thompson 1989, supra.
  • Such techniques can also be utilized to generate T cell subpopulation-related disorder animal models. It should be noted that this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors, e.g. , herpes virus vectors.
  • endogenous target and/or pathway gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target and/or pathway gene (i.e. , the target and/ r pathway gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target or pathway gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the target and/or pathway gene i.e. , the target and/ r pathway gene promoter and/or enhancers
  • the activity of target and/or pathway gene can be reduced using a "dominant negative" approach.
  • constructs which encode defective target and/or pathway gene products can be used in gene therapy approaches to diminish the activity of the target and/or pathway gene product in appropriate target cells.
  • POSITIVE MODULATORY TECHNIQUES As discussed above, successful treatment of certain immune disorders can be brought about by techniques which serve to increase the level of target gene expression or to increase the activity of target gene product, or which, or alternatively, serve to effectively increase the overall number of cells belonging to a specific TH cell subpopulation. As also discussed above, techniques which serve to increase the level of target gene expression or to increase the activity of target gene product can serve to enhance the repair or resolution of ischemia reperfusion injury to an organ or tissue. Thus, such techniques can also be used to successfully treat ischemic disorders or injuries.
  • such molecules can include, but are not limited to proteins or protein fragments of the target gene product, including fragments corresponding to one or more functional domains of the target gene product (e.g., an extracellular domain, a transmembrane domain, or a cytosolic domain) or portions thereof.
  • Such molecules can also include peptides representing soluble extracellular portions of target gene product transmembrane proteins, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof).
  • a compound such as a target gene protein, can, at a level sufficient to ameliorate immune disorder symptoms, be administered to a patient exhibiting such symptoms.
  • Such compounds can also be administered to a patient having an ischemic disorder or injury at a level sufficient to ameliorate the symptoms of the ischemic disorder or injury. Any of the techniques discussed, below, in Section 5.11, can be utilized for such administration.
  • One of skill in the art will readily know how to determine the concentration of effective, non-toxic doses of the compound, utilizing techniques such as those described, below, in Section 5.11.1.
  • fragments or peptides representing a functional domain of a target gene product are administered to an individual at sufficient dosages and such that the fragments or peptides may enhance the target gene product's activity in the individual, e.g., by mimicking the function of the target gene product in vivo .
  • fragments or peptides representing a functional domain of the 200 gene product are administered to a patient which enhance and/or mimic the activity of the endogenous 200 gene product in that patient.
  • Such 200 gene fragments may therefore be used, e.g., to stimulate or increase the repair of injury to tissue or organs resulting from an ischemic injury or disorder.
  • proteins and peptides which may be used in such methods include synthetic (e.g., recombinant or chemically synthesized) proteins and peptides, as well as naturally occurring proteins and peptides.
  • the proteins and peptides may have both naturally occurring and/or non-naturally occurring amino acid residues (e.g., D-amino acid residues) and/or one or more non-peptide bonds (e.g., imino, ester, hydrazide, semicarbazide, and azo bonds) .
  • the proteins or peptides may also contain additional chemical groups (e.g., functional groups) present at the amino and/or carboxy termini, such that, for example, the stability, bioavailability, and/or inhibitory activity of the peptide is enhanced.
  • additional chemical groups e.g., functional groups
  • functional groups include hydrophobic groups (e.g., carbobenzoxyl, dansyl, and t-butyloxycarbonyl groups) an acetyl group, a 9-fluorenylmethoxy-carbonyl group, and macromolecular carrier groups (e.g., lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates) including peptide groups.
  • Suitable dosages and formulations for administration of such peptides and proteins are also well known to those of skill in the art, and are described in Section 5.11 above.
  • the compound to be administered is a peptide compound
  • DNA sequences encoding the peptide compound can be directly administered to a patient exhibiting immune disorder symptoms, at a concentration sufficient to produce a level of peptide compound sufficient to ameliorate the disorder symptoms.
  • Any of the techniques discussed, below, in Section 5.11, which achieve intracellular administration of compounds, such as, for example, liposome administration, can be utilized for the administration of such DNA molecules.
  • the DNA molecules can be produced, for example, by well known recombinant techniques.
  • the DNA molecules encoding such peptides can be taken up and expressed by any cell type, so long as a sufficient circulating concentration of peptide results for the elicitation of a reduction in the immune disorder symptoms, or in a reduction of the symptoms of ischemic disorders and injuries.
  • the DNA molecules encoding such peptides must be taken up and expressed by the TH cell subpopulation of interest at a sufficient level to bring about the reduction of immune disorders.
  • Any technique which serves to selectively administer DNA molecules to the TH cell subpopulation of interest is, therefore, preferred, for the DNA molecules encoding intracellularly acting peptides.
  • techniques for the selective administration of the molecules to TH cell subpopulations residing within lung tissue are preferred.
  • preferred techniques are those which serve to selectively administer DNA molecules to the affected organ or tissue.
  • preferred techniques are those which serve to selectively administer DNA molecules to the affected organ or tissue.
  • ischemic renal disease techniques for the selective administration of the molecules to cells within the kidney are preferred.
  • the TH cell subpopulation-related disorder involves an aberrant gene
  • patients can be treated by gene replacement therapy.
  • One or more copies of a normal target gene or a portion of the gene that directs the production of a normal target gene protein with target gene function can be inserted into cells, using vectors which include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
  • Such gene replacement techniques can be accomplished either in vivo or in vitro.
  • the cell type expressing the target gene is less important than achieving a sufficient circulating concentration of the extracellular molecule for the amelioration of immune disorders, or to ameliorate the symptoms of ischemic disorders or injuries.
  • the gene when the gene encodes a cell which acts intracellularly or as a transmembrane molecule, the gene must be expressed with the TH cell subpopulation cell type of interest. Techniques which select for expression within the cell type of interest are, therefore, preferred for this latter class of target genes. In vivo, such techniques can, for example, include appropriate local administration of target gene sequences.
  • Additional methods which may be utilized to increase the overall level of target and/or pathway gene expression and/or target and/or pathway gene activity include the introduction of appropriate target and/or pathway gene-expressing cells, preferably autologous cells, into a patient at positions and in numbers which are sufficient to ameliorate the symptoms of T cell subpopulation related disorders, or, alternatively, to ameliorate the symptoms of ischemic disorders or injuries.
  • Such cells may be either recombinant or non-recombinant .
  • the cells which can be administered to increase the overall level of target and/or pathway gene expression in a patient are normal cells, which express the target and/or pathway gene.
  • the cells can be administered at the anatomical site of expression, or as part of a tissue graft located at a different site in the body.
  • target gene sequences can be introduced into autologous cells. These cells expressing the target gene sequence of interest can then be reintroduced, preferably by intravenous administration, into the patient such that there results an amelioration of the symptoms of the disorder.
  • TH cells belonging to a specific TH cell subpopulation can be administered to a patient such that the overall number of cells belonging to that TH cell subpopulation relative to other TH cell subpopulation cells is increased.
  • Techniques for such TH cell subpopulation augmentation are described, below, in Section 5.10.3.2.
  • modulatory techniques which, depending on the specific application for which they are utilized, can yield either positive or negative responses leading to the amelioration of immune disorders, including TH cell subpopulation-related disorders.
  • modulatory techniques described in Section 5.10.3.1, below can also be used to treat ischemic disorders and injuries, or, alternatively, to block or inhibit repair of ischemic injuries depending on whether such modulation is positive or negative.
  • the procedures of this Section can be used in conjunction with the negative modulatory techniques described, above, in Section 5.9.1 or, alternatively, in conjunction with the positive modulatory techniques described, above, in Section 5.9.2.
  • ANTIBODY TECHNIQUES Antibodies exhibiting modulatory capability can be utilized to ameliorate immune disorders such as TH cell subpopulation-related disorders, or to treat ischemic disorders and injuries. Depending on the specific antibody, the modulatory effect can be negative and can, therefore, by utilized as part of the techniques described, above, in Section 5.9.1, or can be positive, and can, therefore, be used in conjunction with the techniques described, above, in Section 5.9.2.
  • An antibody having negative modulatory capability refers to an antibody which specifically binds to and interferes with the action of a protein.
  • an extracellular receptor for example, such an antibody would specifically bind the extracellular domain of the receptor in a manner which does not activate the receptor but which disrupts the ability of the receptor to bind its natural ligand.
  • antibodies directed against the extracellular domains of genes 103 or 200 can function as such negative modulators.
  • antibodies directed against one or more of the 10 gene product extracellular domains can function in a negative modulatory manner.
  • Such antibodies can be generated using standard techniques described in Section 5.6, above, against full length wild type or mutant proteins, or against peptides corresponding to portions of the proteins.
  • the antibodies include but are not limited to polyclonal, monoclonal, FAb fragments, single chain antibodies, chimeric antibodies, and the like.
  • An antibody having positive modulatory capability refers to an antibody which specifically binds to a protein and, by binding, serves to, either directly or indirectly, activate the function of the protein which it recognizes.
  • an antibody can bind to the extracellular portion of a transmembrane protein in a manner which causes the transmembrane protein to function as though its endogenous ligand was binding, thus activating, for example, a signal transduction pathway.
  • antibodies can be generated using standard techniques described in Section 5.6, above, against full length wild type or mutant proteins, or against peptides corresponding to portions of the proteins.
  • the antibodies include but are not limited to polyclonal, monoclonal, FAb fragments, single chain antibodies, chimeric antibodies, and the like.
  • the protein such as a target gene protein, to which the antibody is directed is intracellular and whole antibodies are used
  • lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region which binds to the gene product epitope into cells.
  • fragments of the antibody are used, the smallest inhibitory fragment which binds to the protein's binding domain is preferred.
  • peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the protein can be used.
  • Such peptides can be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (e.g.
  • single chain antibodies such as neutralizing antibodies, which bind to intracellular epitopes can also be administered.
  • Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (Marasco, W. et al. , 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893) .
  • any of the administration techniques described, below in Section 5.11 which are appropriate for peptide administration can be 5 utilized to effectively administer the antibodies to their site of action.
  • the separation techniques described herein are based on _ the presence or absence of specific cell surface markers, preferably transmembrane markers.
  • markers can include, but are not limited to, the TH2-specific 103 gene product extracellular domain markers, the THl-specific 200 gene product extracellular domain markers and the TH inducible 10 5 gene product extracellular domain markers.
  • the antibodies used can also be specific to surface markers present on undifferentiated or partially undifferentiated TH cells.
  • the cells can be cultured in physiological buffer or culture medium and induced to differentiate by culturing in the presence of appropriate factors.
  • IL-4 can be added to induce the TH cells to differentiate into TH2 cells
  • the cytokine IL- 12 can be added to induce the TH cells to differentiate into THl cells.
  • cells can be washed, resuspended in, for example, buffered saline, and reintroduced into a patient via, preferably, intravenous administration .
  • Separation techniques can be utilized which separate and purify cells, in vitro, from a population of cells, such as hematopoietic cells autologous to the patient being treated.
  • An initial TH cell subpopulation-containing population of cells, such as hematopoietic cells can be obtained using standard procedures well known to those of skill in the art.
  • Peripheral blood can be utilized as one potential starting source for such techniques, and can, for example, be obtained via venipuncture and collection into heparinized tubes.
  • the T cells such as THl or TH2 cells
  • the T cells can be removed, and thus selectively separated and purified, by various methods which utilize antibodies which bind specific markers present on the T cell population of interest, while absent on other cells within the starting source.
  • These techniques can include, for example, flow cytometry using a fluorescence activated cell sorter (FACS) and specific fluorochromes, biotin-avidin or biotin-streptavidin separations using biotin conjugated to cell surface marker- specific antibodies and avidin or streptavidin bound to a solid support such as affinity column matrix or plastic surfaces or magnetic separations using antibody-coated magnetic beads. Separation via antibodies for specific markers can be by negative or positive selection procedures.
  • FACS fluorescence activated cell sorter
  • specific fluorochromes biotin-avidin or biotin-streptavidin separations using biotin conjugated to cell surface marker- specific antibodies and avidin or streptavidin bound to a solid support such as affinity column matrix or plastic
  • antibodies are used which are specific for markers present on undesired cells.
  • such antibodies could be directed to the extracellular domain of the 200 gene product.
  • TH2 cell subpopulation-related disorders wherein it would be desirable to deplete the number of THl cells
  • such antibodies could be directed to the extracellular domain of the 103 gene product.
  • Cells bound by an antibody to such a cell surface marker can be removed or lysed and the remaining desired mixture retained.
  • antibodies specific for markers present on the desired cells of interest are used which are specific for markers present on the desired cells of interest.
  • THl cell subpopulation-related disorder wherein it would be desirable to increase the number of THl cells
  • such antibodies could be directed to the extracellular domain of the 200 gene product.
  • TH2 cell subpopulation-related disorders wherein it would be desirable to increase the number of THl cells
  • such antibodies could be directed to the extracellular domain of the 103 gene product.
  • Cells bound by the antibody are separated and retained. It will be understood that positive and negative separations can be used substantially simultaneously or in a sequential manner.
  • a common technique for antibody based separation is the use of flow cytometry such as by a florescence activated cell sorter (FACS) .
  • FACS florescence activated cell sorter
  • separation by flow cytometry is performed as follows.
  • the suspended mixture of cells are centrifuged and resuspended in media.
  • Antibodies which are conjugated to fluorochrome are added to allow the binding of the antibodies to specific cell surface markers.
  • the cell mixture is then washed by one or more centrifugation and resuspension steps.
  • the mixture is run through a FACS which separates the cells based on different fluorescence characteristics.
  • FACS systems are available in varying levels of performance and ability, including multi-color analysis.
  • the facilitating cell can be identified by a characteristic profile of forward and side scatter which is influenced by size and granularity, as well as by positive and/or negative expression of certain cell surface markers.
  • Other separation techniques besides flow cytometry can also provide fast separations.
  • One such method is biotin- avidin based separation by affinity chromatography. Typically, such a technique is performed by incubating cells with biotin-coupled antibodies to specific markers, such as, for example, the transmembrane protein encoded by the 103 gene described herein, followed by passage through an avidin column. Biotin-antibody-cell complexes bind to the column via the biotin-avidin interaction, while other cells pass through the column.
  • the specificity of the biotin-avidin system is well suited for rapid positive separation. Multiple passages can ensure separation of a sufficient level of the TH cell subpopulation of interest.
  • the cells derived from the starting source of cells which has now been effectively depleted of TH cell subpopulation cells can be reintroduced into the patient.
  • Such a depletion of the TH cell subpopulation results in the amelioration of TH cell subpopulation-related disorders associated with the activity or overactivity of the TH cell subpopulation.
  • Reintroduction of the TH cell subpopulation-depleted cells can be accomplished by washing the cells, resuspending in, for example, buffered saline, and intravenously administering the cells into the patient.
  • TH cell subpopulation-depleted cells can be reintroduced into patients immediately subsequent to separation.
  • TH cell subpopulation-depleted cells can be cultured and expanded ex vivo prior to administration to a patient. Expansion can be accomplished via well known techniques utilizing physiological buffers or culture media in the presence of appropriate expansion factors such as interleukins and other well known growth factors. In instances whereby the goal of the separation technique is to augment or increase the overall number of cells belonging to a TH cell subpopulation, cells derived from the purified TH cell subpopulation cells can be reintroduced into the patient, thus resulting in the amelioration of TH cell subpopulation-related disorders associated with an under activity of the TH cell subpopulation .
  • the cells to be reintroduced will be cultured and expanded ex vivo prior to reintroduction.
  • Purified TH cell subpopulation cells can be washed, suspended in, for example, buffered saline, and reintroduced into the patient via intravenous administration.
  • Cells to be expanded can be cultured, using standard procedures, in the presence of an appropriate expansion agent which induces proliferation of the purified TH cell subpopulation.
  • an expansion agent can, for example, be any appropriate cytokine, antigen, or antibody.
  • the expansion agent can be IL-4, while for THl cells, the expansion agent can, for example, be IL-12.
  • the purified cells Prior to being reintroduced into a patient, the purified cells can be modified by, for example, transformation with gene sequences encoding gene products of interest. Such gene products should represent products which enhance the activity of the purified TH cell subpopulation or, alternatively, represent products which repress the activity of one or more of the other TH cell subpopulations.
  • Cell transformation and gene expression procedures are well known to those of skill in the art, and can be as those described, above, in Section 5.5.
  • targeting methods can, additionally, be utilized in instances wherein the goal is to deplete the number of cells belonging to a specific TH cell subpopulation.
  • Such targeting methods can be in vivo or in vitro, and can involve the introduction of targeting agents into a population of cells such that the targeting agents selectively destroy a specific subset of the cells within the population. In vivo administration techniques which can be followed for such targeting agents are described, below, in Section 5.11.
  • Targeting agents generally comprise, first, a targeting moiety which, in the current instance, causes the targeting agent to selectively associate with a specific TH cell subpopulation.
  • the targeting agents generally comprise, second, a moiety capable of destroying a cell with which the targeting agent has become associated.
  • Targeting moieties can include, but are not limited to, antibodies directed to cell surface markers found specifically on the TH cell subpopulation being targeted, or, alternatively, to ligands, such as growth factors, which bind receptor-type molecules found exclusively on the targeted TH cell subpopulation.
  • such a targeting moiety can represent an antibody directed against the extracellular portion of the 103 gene product described herein, or can, alternatively, represent a ligand specific for this receptor-type TH2 specific molecule.
  • such a targeting moiety can represent an antibody directed against the extracellular portion of the 200 gene product described herein, or can, alternatively, represent a ligand specific for this receptor-type THl specific molecule.
  • Destructive moieties include any moiety capable of inactivating or destroying a cell to which the targeting agent has become bound.
  • a destructive moiety can include, but it is not limited to cytotoxins or radioactive agents.
  • Cytotoxins include, for example, plant-, fungus-, or bacteria-derived toxins, with deglycosylated Ricin A chain toxins being generally preferred due to their potency and lengthy half-lives.
  • the compounds, nucleic acid sequences and TH cell subpopulation cell described herein can be administered to a patient at therapeutically effective doses to treat or ameliorate immune disorders, e.g. , TH cell subpopulation- related disorders.
  • a therapeutically effective dose refers to that amount of a compound or TH cell subpopulation sufficient to result in amelioration of the immune disorder symptoms of the immune disorder symptoms, or alternatively, to that amount of a nucleic acid sequence sufficient to express a concentration of gene product which results in the amelioration of the TH cell subpopulation-related disorders or of other immune disorders.
  • Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD 50 (the dose lethal to 50% of the population) and the ED 5C (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of antibody, protein, or polypeptide ranges from about 0.001 to 30 mg/kg of body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dose or effective dosage ranges from about 0.001 to 30 mg/kg of body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dose or effective dosage ranges from about 0.001 to 30 mg/kg of body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may also be apparent to one skilled in the art from the results of diagnostic assays as described herein.
  • compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvents can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, subuctaneous, intraperitoneal, intrapulmonary, intranasal, intralesional, vaginal or rectal administration.
  • the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. , pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g.
  • the tablets can be coated by methods well known in the art.
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. , sorbitol syrup, cellulose derivatives or hydrogenated edible fats) ; emulsifying agents (e.g. , lecithin or acacia) ; non-aqueous vehicles (e.g. , almond oil, oily esters, ethyl alcohol or fractionated vegetable oils) ; and preservatives (e.g. , methyl or propyl-p-hydroxybenzoates or sorbic acid) .
  • the preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • compositions for oral administration can be suitably formulated to give controlled release of the active compound.
  • buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner .
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane
  • the compounds can be formulated for parenteral administration (i.e. , intravenous or intramuscular) by injection, via, for example, bolus injection or continuous infusion.
  • parenteral administration i.e. , intravenous or intramuscular
  • Formulations for injection can be presented in unit dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen-free water, before use. It is preferred that the TH cell subpopulation cells be introduced into patients via intravenous administration.
  • the compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g. , containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds can also be formulated as a depot preparation.
  • Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) 5 or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble 0 salt.
  • Intralesional administration can comprise, for example, perfusing or contacting a graft or organ with a composition prior to transplantation.
  • compositions can, if desired, be presented in a pack 5 or dispenser device which can contain one or more unit dosage forms containing the active ingredient.
  • the pack can for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration. 0
  • Antibodies which specifically bind to target gene products of the invention and thereby modulate their activity _ can also be administered to a patient at therapeutically effective doeses to treat or ameliorate immune disorders, or to treat or ameliorate ischemic disorders or injuries.
  • Section 13, below demonstrates the use of anti-200 gene product antibodies to block recovery of kidney tissue _ from ischemia reperfusion injury.
  • Antibodies of the invention are administered by any suitable means, including those described in Section 5.12.2, above.
  • antibody to a target gene product of the invention is suitably administered by pulse infusion, 5 particularly with declining doses of the antibody.
  • the dosing is administered by injections, most preferably by intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody may suitably administered to the patient at one time or, more preferably, over a series of treatments.
  • the initial pharmaceutically effective amount of antibody administered parenterally will be in the range of about 0.1 to 20 mg/kg of patient body weight per day, with the typical initial range being 0.3 to 15 mg/kg of patient body weight per day.
  • the subsequent dosing is less than 100% of initial dosing, such subsequent dosing is calculated on the basis of daily dosing.
  • the subsequent dosing is less than about 50%, more preferably, less than about 25%, still more preferably, less than about 10%, and still more preferably, less than about 5%. Most preferably, the subsequent dosing is less than about 2% of the initial dosing.
  • dosage ranges to be administered will, preferably, range from about 1 ⁇ g/kg to about 100 mg/kg, 1 ⁇ g/kg to about 15 mg/kg, or about 1 ⁇ g/kg, to about 2.0 mg/kg body weight.
  • the preferred scheduling is that the initial dosing is administered no less frequently than daily, and up to an including continuously by infusion. More preferably, depending on the specific disorder or injury, the initial daily dosing is administered for at least about one week, and preferably at least about two weeks. To obtain the most efficacious results, the inital dosing is preferably given as close to the first sign, diagnosis, appearance, or occurrence of the disorder as possible, or, particular in the case of immune disorders, during remission of the disorder. Subsequent dosing is preferably administered periodically no more about once a week. More preferably, the subsequent dosing is administered no more than once biweekly. Subsequent dosing is typically administered for at least about five weeks, and preferably for at least about 10 weeks, after the initial dosing is terminated.
  • DIAGNOSTIC AND MONITORING TECHNIQUES A variety of methods can be employed for the diagnosis of immune disorders, e.g.. TH cell subpopulation-related disorders, predisposition to such immune disorders, for monitoring the efficacy of anti-immune disorder compounds during, for example, clinical trials and for monitoring patients undergoing clinical evaluation for the treatment of such disorders. Further, a number of methods can be utilized for the detection of activated immune cells, e.g. , activated members of TH cell subpopulations.
  • Such methods can, for example, utilize reagents such as the fingerprint gene nucleotide sequences described in Sections 5.1, and antibodies directed against differentially expressed and pathway gene peptides, as described, above, in
  • Such reagents can be used, for example, for: 1) the detection of the presence of target gene expression, target gene mutations, the detection of either over- or under-expression of target gene mRNA relative to the non-immune disorder state or relative to an unactivated TH cell subpopulation; 2) the detection of either an over- or an underabundance of target gene product relative to the non-immune disorder state or relative to the unactivated TH cell subpopulation state; and 3) the identification of specific TH cell subpopulation cells (e.g. , TH cells involved in an immune disorder, or activated TH cells) within a mixed population of cells.
  • specific TH cell subpopulation cells e.g. , TH cells involved in an immune disorder, or activated TH cells
  • the methods described herein can be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific fingerprint gene nucleic acid or anti- fingerprint gene antibody reagent described herein, which can be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting THl- or TH2-related abnormalities.
  • Any cell type or tissue, preferably TH cells, in which the fingerprint gene is expressed can be utilized in the diagnostics described below.
  • methods for monitoring the efficacy of compounds in clinical trials for the treatment of immune disorders can, for example, be compounds such as those described, above, in Section 5.9.
  • Such a method comprises detecting, in a patient sample, a gene transcript or gene product which is differentially expressed in a TH cell subpopulation in an immune disorder state relative to its expression in the TH cell subpopulation when the cell subpopulation is in a normal, or non-immune disorder, state.
  • nucleic acid detection techniques described, below, in Section 5.12.1 or any of the peptide detection techniques described, below, in Section 5.12.2 can be used to detect the gene transcript or gene product which is differentially expressed in the immune disorder TH cell subpopulation relative to its expression in the normal, or non-immune disorder, state.
  • the expression of a single fingerprint gene, or alternatively, the fingerprint pattern of a TH cell subpopulation can be determined for the TH cell subpopulation in the presence or absence of the compound being tested.
  • the efficacy of the compound can be followed by comparing the expression data obtained to the corresponding known expression patterns for the TH cell subpopulation in a normal, non-immune disorder state.
  • Compounds exhibiting efficacy are those which alter the single fingerprint gene expression and/or the fingerprint pattern of the immune disorder TH cell subpopulation to more closely resemble that of the normal, non-immune disorder TH cell subpopulation.
  • the detection of the product or products of genes differentially expressed in a TH cell subpopulation in an immune disorder state relative to their expression in the TH cell subpopulation when the cell subpopulation is in a normal, or non-immune disorder, state can also be used for monitoring the efficacy of potential anti-immune disorder compounds during clinical trials.
  • the level and/or activity of the products of one or more such differentially expressed genes can be determined for the TH cell subpopulation in the presence or absence of the compound being tested.
  • the efficacy of the compound can be followed by comparing the protein level and/or activity data obtained to the corresponding known levels/activities for the TH cell subpopulation in a normal, non-immune disorder state.
  • Compounds exhibiting efficacy are those which alter the pattern of the immune disorder TH cell subpopulation to more closely resemble that of the normal, non-immune disorder TH cell subpopulation.
  • the detection of 103 gene transcripts and/or products can be particularly suitable for monitoring the efficacy of compounds in clinical trials for the treatment of TH2 cell subpopulation-related immune disorders such as, for example, asthma or allergy.
  • the expression patterns of the 105, 106 and 200 genes in THl cell subpopulations relative to TH2 cell subpopulations can make the detection of transcripts and/or products of these genes particularly suitable for monitoring the efficacy of compounds in clinical trials for the treatment of THl cell subpopulation-related immune disorders such as, for example, multiple sclerosis, psoriasis or insulin dependent diabetes.
  • TH cell responsiveness for example, responsiveness to antigen
  • activated immune cells e.g. , activated members of TH cell subpopulations.
  • Detection methods such as these are important in that many immune disorders involve inappropriate rather than insufficient immune responses.
  • detection methods can be used, for example, to detect a predisposition to an immune disorder.
  • Methods for detecting TH cell responsiveness and/or activation can comprise, for example, detecting in a TH cell sample a gene transcript or product which is differentially expressed in TH cell subpopulation which is in an activated or responsive state (e.g. , a state in which the TH cell subpopulation has been exposed to antigen) , relative to a TH cell subpopulation which is in an unactivated or nonresponsive state.
  • an activated or responsive state e.g. , a state in which the TH cell subpopulation has been exposed to antigen
  • Any of the nucleic acid detection techniques described, below, in Section 5.12.1 or any of the peptide detection techniques described, below, in Section 5.12.2 can be used to detect such a differentially expressed gene transcript or gene product.
  • such techniques can also be utilized as part of methods for identifying compounds which alter the cellular expression of one or more of the differentially expressed genes described herein, or as part of methods for identifying compounds which alter the cellular and/or secreted level of product produced by the differentially expressed genes described herein.
  • such techniques can be used to identify compounds which alter the level of expression of the 103 gene or the level of 103 gene product present in a cell.
  • Such methods can include, for example, contacting a T cell with a compound, measuring the level of 103 gene expression in the cell (or the level of 103 gene product in the cell) , then comparing the level obtained to that of a cell not exposed to the compound.
  • the T cells used herein can include, for example, THO, THl or TH2 cells.
  • Such methods can furthe include stimulating the cells, for example, stimulating the cells prior to contacting the cells with the compound. Among the methods for stimulation are stimulation via anti-CD3 antibody stimulation.
  • Such methods can be performed such that the cell contacted is presented within a non-human mammal, for example, a mouse.
  • a non-human mammal for example, a mouse.
  • the non-human mammals which can be utilized as part of these methods are ones which exhibit symptoms of a T cell-related disorder (such as, for example a TH2-related disorder, e.g. , asthma) , and contacting the cell with the compound can ameliorate symptoms of the disorder.
  • a T cell-related disorder such as, for example a TH2-related disorder, e.g. , asthma
  • the TH2-specific nature of the 103 gene can make the detection of its gene transcripts and/or products particularly suitable for detecting activation and/or responsiveness of TH2 cells. Further, the THl-specific nature of the 105, 106 and 200 genes can make the detection of transcripts and/or products of these genes particularly suitable for the detection of THl activation and/or responsiveness .
  • DNA or RNA from the cell type or tissue to be analyzed can easily be isolated using procedures which are well known to those in the art. Diagnostic procedures can also be performed "in situ” directly upon, for example tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents such as those described in Section 5.4 can be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, "PCR In Situ Hybridization: Protocols and Applications", Raven Press, NY). Expression of specific cells within a population of cells can also be determined, via, for example, in situ techniques such as those described above, or by standard flow cytometric techniques.
  • Fingerprint gene nucleotide sequences can, for example, be used in hybridization or amplification assays of biological samples to detect TH cell subpopulation-related disorder gene structures and expression.
  • assays can include, but are not limited to, Southern or Northern analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses can reveal both quantitative aspects of the expression pattern of the fingerprint gene, and qualitative aspects of the fingerprint gene expression and/or gene composition.
  • Such techniques can detect not only the presence of gene expression, but can also detect the amount of expression, particularly which specific cells are expressing the gene of interest, and can, further, for example, detect point mutations, insertions, deletions, chromosomal rearrangements, and/or activation or inactivation of gene expression.
  • Diagnostic methods for the detection of fingerprint gene-specific nucleic acid molecules can involve for example, contacting and incubating nucleic acids, derived from the cell type or tissue being analyzed, with one or more labeled nucleic acid reagents as are described in Section 5.4, under conditions favorable for the specific annealing of these reagents to their complementary sequences within the nucleic acid molecule of interest.
  • the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides.
  • all non-annealed nucleic acids are removed from the nucleic acid: fingerprint molecule hybrid.
  • the presence of nucleic acids from the cell type or tissue which have hybridized, if any such molecules exist, is then detected.
  • the nucleic acid from the tissue or cell type of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
  • non- annealed, labeled nucleic acid reagents of the type described in Section 5.4 are easily removed. Detection of the remaining, annealed, labeled fingerprint nucleic acid reagents is accomplished using standard techniques well-known 5 to those in the art.
  • a cDNA molecule is obtained from an RNA molecule of interest (e.g. , by reverse transcription of the RNA molecule into cDNA) .
  • RNA Ribonucleic acid sequence
  • TH0, THl and/or TH2 cell type-containing tissues A sequence within the cDNA is then used as the template for a nucleic acid sequence
  • nucleic acid reagents used as synthesis initiation reagents (e.g. , primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the fingerprint gene nucleic
  • the preferred lengths of si ⁇ h nucleic acid reagents are at least 9-30 nucleotides.
  • the nucleic acid amplification can be performed using radioactively or non-radioactively labeled nucleotides. Alternatively, enough amplified product can be made such that the product can be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
  • Fingerprint patterns in this context, contain the pattern of mRNA expression of a series (i.e. , at least two and up to the total number present) of fingerprint genes obtained for a given tissue or cell type under a given set of conditions.
  • Such conditions can include, for example, TH cell subpopulation-related disorders, and conditions relevant to processes involved in the differentiation, maintenance and effector function of TH cell subpopulations.
  • THl-related disorders can include, for example, chronic inflammatory diseases and disorders, such as Crohn ' s disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific autoimmunity, including multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, graft rejection, graft versus host disease and sarcoidosis.
  • TH2-related disorders can include, for example, atopic conditions, such as asthma and allergy, including allergic rhinitis, gastrointestinal allergies, including food allergies, eosinophilia, conjunctivitis, glomerular nephritis, certain pathogen susceptibilities such as helminthic (e.g.
  • Fingerprint patterns can be generated, for example, by utilizing a differential display procedure, as discussed, above, in Section 5.1.1.2, Northern analysis and/or RT-PCR. Any of the gene sequences described, above, in Section 3.2.1 can be used as probes and/or RT-PCR primers for the generation and corroboration of such fingerprint patterns. 5.12.2.
  • Antibodies directed against wild type or mutant fingerprint gene peptides can also be used as TH cell subpopulation- related disorder diagnostics and prognostics, as described, for example, herein.
  • diagnostic methods can be used to detect fingerprint gene product, abnormalities in the level of fingerprint gene protein expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of fingerprint gene protein.
  • Structural differences can include, for example, differences in the size, electronegativity, or antigenicity of the mutant fingerprint gene protein relative to the normal fingerprint gene protein.
  • Protein from the tissue or cell type to be analyzed can easily be isolated using techniques which are well known to those of skill in the art.
  • the protein isolation methods employed herein can, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D. , 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
  • Preferred diagnostic methods for the detection of wild type or mutant fingerprint gene peptide molecules can involve, for example, immunoassays wherein fingerprint gene peptides are detected by their interaction with an anti- fingerprint gene product-specific antibody.
  • antibodies, or fragments of antibodies, such as those described, above, in Section 5.6, useful in the present invention can be used to quantitatively or qualitatively detect the presence of wild type or mutant fingerprint gene peptides.
  • This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below, this Section,) coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • a fluorescently labeled antibody see below, this Section,
  • fluorimetric detection are especially preferred if the fingerprint gene peptides are expressed on the cell surface, such as, for example, is the case with the 10 gene product, the 200 gene product and the transmembrane form of 103 gene product.
  • the techniques described herein can be used to detect specific cells, within a population of cells, which express the fingerprint gene product of interest.
  • the antibodies (or fragments thereof) useful in the present invention can, additionally, be employed histologically , as in immunofluorescence or immunoeiectron microscopy, for in situ detection of fingerprint gene peptides.
  • In situ detection can be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays for wild type or mutant fingerprint gene peptides typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying fingerprint gene peptides, and detecting the bound antibody by any of a number of techniques well-known in the art.
  • a biological sample such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture
  • the biological sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support can then be washed with suitable buffers followed by treatment with the detectably labeled fingerprint gene-specific antibody.
  • the solid phase support can then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on solid support can then be detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface can be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • the binding activity of a given lot of anti-wild type or mutant fingerprint gene product antibody can be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • One of the ways in which the fingerprint gene peptide-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA)
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection can also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography .
  • the antibody can also be labeled with a fluorescent compound.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound can be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • T cell paradigm described, above, in Section 5.1.1.1, was utilized to identify a gene, designated herein as the 102 gene, which is expressed in TH2 cells.
  • the identified gene is present in TH2 cells at a much higher level than in THl cells.
  • the Example presented herein demonstrates the usefulness of the paradigm approach of the invention for the identification of genes that are differentially expressed in
  • TH cell subpopulations TH cell subpopulations. 6.1. MATERIALS AND METHODS Transgenic mice: Naive CD4 + cells were obtained from the spleens and/or lymph nodes of unprimed transgenic mouse strains harboring a T cell receptor (TCR) recognizing ovalbumin (Murphy et al., 1990, Science 250: 1720) .
  • TCR T cell receptor
  • Ova-specific transgenic T cells Suspensions of ova- specific T cells were co-cultured with stimulatory peptide antigen and antigen presenting cells essentially as described in Murphy et al. (Murphy et al., 1990, Science 250: 1720) . Briefly, 2-4 x 10 6 T cells were incubated with approximately twice as many TA3 antigen presenting cells in the presence of 0.3 ⁇ M Ova peptide. THl cultures contained approximately 10 ng/ml recombinant mIL-12. Conversely, TH2 cells received IL- 4 (lOOOu/ml) . Cultures were harvested at various time points after initiation of culture.
  • T cells were purified of TA3 cells using anti-CD4 coated magnetic beads (Dynal, Inc.). T cells were pelleted by gentle centrifugation and lysed in the appropriate volume of RNAzolTM (Tel-Test, Friendswood, TX) .
  • Tissue collection and RNA isolation Cells were quick frozen on dry ice. Samples were then homogenized together with a mortar and pestle under liquid nitrogen.
  • RNAzolTM or RNAzolBTM Total cellular RNA was extracted from tissue with either RNAzolTM or RNAzolBTM (Tel-Test, Friendswood, TX) , according to the manufacturer's instructions. Briefly, the tissue was solubilized in an appropriate amount of RNAzolTM or RNAzolBTM, and RNA was extracted by the addition of 1/10 v/v chloroform to the solubilized sample followed by vigorous shaking for approximately 15 seconds. The mixture was then centrifuged for 15 minutes at 12,000g and the aqueous phase was removed to a fresh tube. RNA was precipitated with isopropanol. The resultant RNA pellet was dissolved in water and re-extracted with an equal volume of chloroform to remove any remaining phenol. The extracted volume was precipitated with 2 volumes of ethanol in the presence of 150mM sodium acetate. The precipitated RNA was dissolved in water and the concentration determined spectroscopically (A 260 ) .
  • RNA Total cellular RNA (10-50 ⁇ g) was treated with 20 Units DNase I (Boehringer Mannheim, Germany) in the presence of 40 Units ribonuclease inhibitor (Boehringer Mannheim, Germany) . After extraction with phenol/chloroform and ethanol precipitation, the RNA was dissolved in DEPC (diethyl pyrocarbonate) -treated water. Differential mRNA display was carried out as described, above, in Section 5.1.1.2. RNA (0.4-2 ⁇ g) was reverse- transcribed using Superscript reverse transcriptase (GIBCO/BRL) . The cDNAs were then amplified by PCR on a Perkin-Elmer 9600 thermal cycler.
  • DNase I Boehringer Mannheim, Germany
  • ribonuclease inhibitor Boehringer Mannheim, Germany
  • the reaction mixtures (20 ⁇ l) included arbitrary decanucleotides and one of twelve possible T 1X VN sequences, wherein V represents either dG, dC, or dA, and N represents either dG, dT, dA, or dC.
  • Parameters for the 40 cycle PCR were as follows: Hold 94° C. 2 minutes; Cycle 94° C. 15 seconds, 40° C. 2 minutes; Ramp to 72° 30 seconds; Hold 72° C. 5 minutes; Hold 4° C.
  • Radiolabelled PCR amplification products were analyzed by electrophoresis on 6% denaturing polyacrylamide gels.
  • PCR bands of interest were recovered from sequencing gels and reamplified.
  • Pellets were resuspended in lO ⁇ l distilled water. 5 ⁇ l of the eluted DNA were reamplified in a lOO ⁇ l reaction containing: standard Cetus Taq polymerase buffer, 20 ⁇ M dNTPs, l ⁇ M of each of the oligonucleotide primers used in the initial generation of the amplified DNA. Cycling conditions used were the same as the initial conditions used to generate the amplified band, as described above. One-half of the amplification reaction was run on a 2% agarose gel and eluted using DE-81 paper (Whatman Paper, Ltd. , England) as described in Sambrook et al., supra .
  • Recovered fragments were ligated into the cloning vector pCR tm II (Invitrogen, Inc., San Diego CA) and transformed into competent E. coli strain DH5 (Gibco/BRL, Gaithersburg, MD) . Colonies were grown on LB-agar plates containing ampicillin (lOO ⁇ g/ml) and X-gal (40 ⁇ g/ml) to permit blue/white selection.
  • RNA samples were electrophoresed in a denaturing agarose gel containing 1-1.5% agarose (SeaKemTM LE, FMC BioProducts, Rockland, ME) containing 6.3% formaldehyde. Samples containing 5-2O ⁇ g of total RNA were mixed with denaturing loading solution (72% deionized formamide and bromophenol blue) and heated to 70°C. for 5 minutes. Samples were placed on ice and immediately loaded onto gels. Gels were run in lx MOPS buffer (lOOmM MOPS, 25mM sodium acetate, 5mM EDTA) . After electrophoresis, the gels were stained with ethidium bromide and visualized with ultraviolet light.
  • lx MOPS buffer lOOmM MOPS, 25mM sodium acetate, 5mM EDTA
  • RNA was soaked in 50mM sodium hydroxide with gentle agitation for approximately 30 minutes to lightly cleave RNA. Gels were rinsed twice in water and then neutralized by soaking in 0.1M Tris-HCl (pH 7.5) for approximately 30 minutes. Gels were briefly equilibrated with 2Ox SSC (3M sodium chloride, 0.3M sodium citrate) and then transferred to nylon membranes such as HybondTM,-N, (Amersham, Inc., Arlington Heights, IL) or Zeta- Probe (Bio-Rad, Inc., Hercules, CA) overnight in 20x SSC. Membranes containing transferred RNA were baked at 80°C. for 2 hours to immobilize the RNA.
  • 2Ox SSC 2Ox SSC
  • DNA fragments to be used as probes were of various sizes and were labeled using a random hexamer labeling technique. Briefly, 25ng of a purified DNA fragment was used to generate each probe. Fragments were added to a 20 ⁇ l random hexanucleotide labeling reaction (Boehringer Mannhein, Inc. , Indianapolis, IN) containing random hexamers and a mix of the nucleotides dCTP, dGTP, and dTTP (at a final concentration of 25 ⁇ M each) . The reaction mix was heat-denatured at 100°C. for 10 minutes and then chilled on ice.
  • Nylon membranes containing immobilized RNA were prehybridized according to manufacturer's instructions. Radiolabelled probes were heat denatured at 70°C. in 50% deionized formamide for 10 minutes and ten added to the hybridization mixture (containing 50% formamide, 10% dextran sulfate, 0.1% SDS, lOO ⁇ g/ml sheared salmon sperm DNA, 5x SSC, 5x Denhardt's solution, 30mM Tris-HCl (pH 8.5), 50mM NaP0 4 (pH 6.5). Hybridizations were carried out at 42°C. overnight.
  • Nylon membranes were then bathed for 2 minutes in a wash solution of 0.2x SSC and 0.1% SDS at room temperature to remove most of the remaining hybridization solution. The membranes were then bathed twice in fresh 42°C. preheated wash solution for 20 minutes. Filters were covered in plastic wrap and exposed to autoradiographic film to visualize results.
  • FIG. 1 shows amplified fragments obtained from these samples, with the arrow indicating a PCR product, designated band 102, which was judged to represent a cDNA derived from RNA produced by a gene which is expressed at a higher level in TH2 cell subpopulations, relative to THl cell subpopulations.
  • the gene corresponding to band 102 is referred to herein as the 102 gene.
  • the amplified band 102 cDNA was recovered, reamplified, subcloned into a cloning vector and sequenced, as described, above, in Section 6.1.
  • a BLAST Altschul, S.F. et al., 1990, J. Mol. Biol. 215: 403-410) database search with this consensus sequence resulted in an alignment with 98% identity to the mouse Granzyme A, or Hanukah factor, gene, (Masson, D. et al., 1986, FEBS Lett. 208:84-88; Masson, D.
  • amplified band 102 cDNA was used to probe Northern RNA blots containing RNA samples from THl and TH2 cell lines, and from spleen and thymus tissue.
  • FIG. 3 shows the results of one such Northern blot analysis, in which the steady state level of message for 102 gene mRNA are significantly increased in RNA samples derived from stimulated TH2 versus THl samples. Further, the positive signals in both thymus and spleen RNA samples supports the indication that the 102 gene product is involved in some aspect of T cell function.
  • the Northern analysis confirmed the putative differential TH2 regulation which had been suggested by the differential display result.
  • a TH2 differentially regulated gene designated here the 102 gene, and corresponding to the mouse Granzyme A/Hanukah factor gene, has been identified, thereby corroborating the usefulness of such paradigms in identifying genes expressed preferentially in T helper cell subpopulations such as THl or TH2 cell populations.
  • the gene identified here had previously been found to be expressed in natural killer T cells and, further, had been reported to be expressed in a fraction of CD4 + cells (Fruth, U. et a;., 1988, Eur. J. Imm. 18:773-781; Liu, C.C. et al., 1989, J. Exp. Med. 170:2105-2118), the results described herein represent the first instance in which a TH cell subpopulation role for this gene has been found. Prior to this study, the gene had been reported to be expressed in T cells in a variety of situations, including THl cell subpopulation- and TH2 cell subpopulation-related disorders.
  • Granzyme A/Hanukah factor expression has been reported in allograft rejection (Muller, C. et al., 1988, J. Exp. Med. 167 : 1124-1136) and autoimmune diseases (Ojcius, D.M. and Young, D.E., 1990, Cancer Cells 2:138-145; Young, L.H.Y. et al., 1992, Am. J. Path. 140:1261-1268) , which are THl cell subpopulation-related disorders, and also in Leishmania infection susceptible mice (Moll, H. et al., 1991, Inf. and Imm. 59:4701-4705) and in leprosy lesions 5 (Ebnet, K. et al., 1991, Int. Imm. 2:9-19; Cooper, C.L. et al., 1989, J. Exp. Med. 169:1565-1581), which are both TH2 cell subpopulation-related disorders.
  • the differential TH2-like expression demonstrated here represents, therefore, the first molecular evidence clearly 0 indicating a more primary role for the gene product in TH2 versus THl cell subpopulations.
  • SPECIFIC GENE 5 In the Example presented in this Section, the transgenic T cell paradigm, described, above, in Sections 5.1.1.1 and 6, was utilized to identify a gene which is differentially expressed in TH2 cells. Specifically, this gene is present in TH2 cells while being completely absent from THl cells. _ The gene, which corresponds to a gene known, alternatively, as ST-2, Tl and Fit-1, does not appear to be expressed in any other assayed cell type or tissue, and is demonstrated here for the first time to encode a marker which is, in vivo, completely TH2-specific. The 103 gene encodes a cell surface 5 protein, the potential significance of which is discussed herein.
  • Expression levels were determined by PCR amplification of serial dilutions of first strand cDNA.
  • cDNA is serially diluted in water.
  • the dilutions are then batch amplified by PCR using sequence-specific primers. All PCR reactions are amplified under identical conditions. Therefore, the amount of product generated should reflect the amount of sequence template which was initially present.
  • b- 10 fold dilutions of cDNA were used and enough dilutions were used such that the amount of product subsequently produced ranged from clearly visible, by UV illumination of ethidium bromide-stained gels, to below detection levels. The method described herein can distinguish 10-fold differences in expression levels.
  • Primers were designed for the amplification of the sequenced amplified bands, which were chosen using the program OLIGO (National Biosciences, Plymouth, MN) .
  • Primer sequences used in this assay were as follows: and 103 sense primer, 5 ' -TTGCCATAGAGAGACCTC-3 ' (SEQ ID NO: 18); band 103 antisense primer, 5 '-TGCTGTCCAATTATACAGG-3 ' (SEQ ID NO: 19); murine gamma actin sense primer, 5 ' -GAACACGGCATTGTCACTAACT-3 ' (SEQ ID NO: 20); murine gamma actin antisense primer, 5'- CCTCATAGATGGGCACTGTGT-3 ' (SEQ ID NO: 21).
  • RNAse protection assays were performed according to manufacturer's instructions, using a kit purchased from Ambion, Inc.
  • RNA probes derived from GenBank Accession No. Y07519 were utilized in the RNAse protection assays. These probes were also generated according to manufacturer's instructions, using a kit purchased from Ambion, Inc. The sequence of these RNA probes corresponds to the 5' end of the gene, and includes both coding and 5' untranslated sequences.
  • Anti CD-3 stimulation Conditions were as described, below, in Section 8.1.
  • RNA isolated from THl and TH2 cell samples obtained from a transgenic T cell paradigm study as described, above, in Section 6.1. Specifically, TH cells were obtained from transgenic mice harboring a T cell receptor recognizing ovalbumin (Murphy et al., 1990, Science 250: 1720) were stimulated three times, and RNA was obtained from THl and TH2 cells. Differential display analysis of the RNA samples resulted in the identification of a TH2 differentially expressed band, designated and referred to herein as band 103. The gene corresponding to band 103 is referred to herein as the 103 gene.
  • nucleotide sequence of band 103 resulted in an alignment with 98% identity to the mouse form of a gene known, alternatively, as the ST-2 , Tl or Fit-1 gene (Klemenz, R. et al., 1989, Proc. Natl. Acad. Sci. USA 86:5708-5712; Tominaga, S., 1989, FEBS Lett. 258:301-301; Werenskiold, A.K. et al., 1989, Mol. Cell.
  • the 103 gene encodes, possibly via alternatively spliced transcripts, transmembrane and soluble forms of proteins which belong to the immunoglobulin superfamily.
  • the soluble form of the protein shows a high level of similarity to the 5 extracellular portion of the mouse interleukin-1 receptor type 1 (IL-1R1) and interleukin-1 receptor type 2 (IL-1R2; which lacks a cytoplasmic domain) , while the transmembrane portion (termed ST2L) bears a high resemblance to the entire IL-1R1 sequence and to the extracellular IL-1R2 sequences.
  • IL-1R1 interleukin-1 receptor type 1
  • IL-1R2 interleukin-1 receptor type 2
  • the 103 gene appears to be tightly linked to the interleukin 1 receptor-type 1 locus (McMahan, C.J. et al., 1991, EMBO J. 10:2821-2832; Tominaga, S. et al., 1991, Biochem. Biophys. Acta. 1090: 1-8) . Additionally, the human 103 gene homolog has also been reported (Tominaga, S. et al.,
  • FIG. 4B illustrates the 103 gene transmembrane and soluble forms of protein, and shows their relationship to the IL-1R1 protein sequence.
  • TH2 specificity of the 103 gene was further confirmed by a Northern analysis of several representative TH cell lines. Specifically, three TH2 clones (CDC25, D10.G4, DAX) and three THl clones (AE7.A, Dorris, Dl.l) were utilized
  • RNA samples were isolated from either unstimulated cells or from cells which had been stimulated for 6 hours with plate-bound anti-CD3 antibody. The samples were probed with band 103 sequences, as shown in FIG. 6. While 103 gene RNA is present in RNA obtained from both unstimulated and
  • 103 gene RNA is completely absent from all of the samples obtained from either stimulated or unstimulated THl cells. As the RT-PCR analysis described above first demonstrated, the 103 gene appears to be TH2 specific, with no detectable THl-derived signal being present.
  • the data presented in FIG. 7 represent an additional Northern analysis in which 103 gene expression was assayed in TH cell clones (lanes 1-5) and in murine tissues (lanes 6- 10) .
  • the data presented here demonstrate that 103 gene expression appears to be negative in each of the tissues (i.e. , brain, heart, lung, spleen, and liver) tested.
  • FIG. 8 illustrates an RNAse protection assay which demonstrates two points regarding 103 gene regulation.
  • this analysis of TH cell clones confirms the TH2- specific results described, above. Specifically, the results of this study demonstrate by RNase protection, that 103 gene mRNA is absent from the THl clone AE7 , but is present in the TH2 clone D10.G4.
  • RNAse protection revealed that alternate forms of 103 gene transcripts are produced upon stimulation of TH2 clones. Specifically, within 6 hours of anti-CD3 stimulation, two additional forms of 103 gene transcript appear in TH2 clones. These additional 103 gene transcript forms represent, one, a transcript encoding a shortened, secreted, soluble form of the band 103 gene product, and, two, a smaller, termed mini, transcript which encodes a yet shorter form of the gene product. Thus, it appears that, while the 103 gene transcript encoding the transmembrane gene product is expressed in both unstimulated and stimulated TH2 cells, the two shorter forms of transcript are expressed in a TH2-specific inducible manner.
  • the 103 gene transcript encoding the transmembrane product are expressed in both stimulated and unstimulated TH2 cells, the level of this transcript present in stimulated is lower, i.e. , is downregulated.
  • the lower level of transmembrane product and higher level of secreted 103 gene product- can act synergistically to dampen some stimulation-induced signal transduction event.
  • results presented herein represent the first time the mini form of 5 103 gene transcript, which can encode a shorter version of the soluble form of 103 gene product, has been observed.
  • the full length 103 gene product can be utilized in a variety of methods to modulate TH cell subpopulation-related disorders and/or to 0 identify compounds which exhibit such modulatory capability.
  • the truncated forms of the 103 gene products can, additionally, be used as part of these methods. Modulatory methods are described, above, in Section 5.9, while strategies for the identification of modulatory compounds are 5 described, above, in Section 5.8.
  • T cell clone paradigm searches were conducted as described, above, in Section 5.1.1.1. Specifically, the TH cell clone paradigms used three different clones: D10.G4 (TH2), AE7 (THl) and Dl.l (THl). Prior to stimulation, cell cultures were enriched for live cells by centrifugation through a Ficoll gradient. Recovered cells were counted and their viability was examined using trypan blue exclusion. Cells were replated into either T25 or T75 flasks at approximately 5 x 10 6 cells in 5 mis or 1.5 x 10 s cells in 10 mis of culture medium, respectively.
  • Coating was performed, generally, according to Current Protocols in Immunology, 1992, Coligan, J.E. et al., John Wiley & Sons, NY, pp 3.12.4-3.12.6). Specifically, prior to plating, the flasks were coated with anti-CD3-e antibodies (hybridoma supernatant from the 145-C11 hybridoma; Purngen, Inc., San Diego CA) . For coating, antibodies were resuspended in PBS at 1-2 ⁇ g/ml at a volume sufficient to coat the bottom of the flasks. Coating solution was incubated on the flasks for at least one hour at 37° C.
  • the THl cDNA library was constructed using a Gibco BRL SuperscriptTM Lambda System Kit, according to manufacturer's instructions. Briefly, 4.5 ⁇ g of purified mRNA was used as starting material for the synthesis of poly A-primed first strand cDNA containing a Not-1 cloning site. Second strand cDNA synthesis was performed with RNAse H treatment followed by random priming. Sal-1 adaptors were ligated to the 5' end of the resulting double-stranded cDNA. The ligated cDNA was digested with Not-1 and size fractionated.
  • the cDNA library was screened with a probe generated by labeling the entire sequence of the band 200 subclone O, which was constructed using amplified DNA obtained from the differential display analysis.
  • the band 200 sequence was excised from the pCRII Cloning VectorTM (Invitrogen) by digestion with EcoRI. Approximately 1/100,000 cDNA library plaques were scored as positive when screened with this probe. Several clones, including 200-P and 20OAF, were chosen for further study.
  • the cDNA library described above was also used to isolate 54 gene cDNA clones. For screening, the entire excised band 54 insert was used as a probe.
  • Transgenic T cell paradigm and T cell clone paradigm searches were conducted to identify gene sequences which represent genes differentially expressed within and/or among TH cell subpopulations and/or during the differentiation of such subpopulations. Described herein are several novel genes which have been identified via these paradigm searches. Specifically, the genes described herein have been designated the 10, 54, 57, 105, 106, 161 and 200 genes. A summary of the differential expression characteristics of the novel gene sequences described herein is presented in Table 1, above.
  • the band 10 and 57 have been identified as TH inducible gene sequences. That is, the expression of such genes in unstimulated TH cells is either undetectable or is detectable at extremely low levels, but is upregulated in both stimulated THl and TH2 cells. In fact, the 10 gene expression is detectable as early as 6 hours post stimulation. Thus, such gene products can be involved in the activation of TH cells and/or can be involved in the maintenance of mature TH cell function, in a non-TH cell subpopulation-specific manner.
  • FIG. 9 depicts the nucleotide sequence (SEQ ID NO: 3) of the 10 gene coding region and the derived amino acid sequence of the 10 gene product (SEQ ID NO: 10). While database searches reveal that the 10 gene sequence is novel, that is, has not previously been reported in the databases, an analysis of the portion of the 10 gene corresponding to the band 10 nucleotide sequence (the underlined portion of the nucleotide sequence of FIG. 9) shows, as depicted in FIG. 10A-C, a high similarity to a specific class of known gene products. Specifically, as the hydrophilicity plots of FIG. 10A-C show, the 10 gene product appears to encode a protein having a seven transmembrane domain sequence motif.
  • the gene products belonging to this class of protein tend to represent G protein-coupled receptor molecules.
  • the TH inducible expression of the 10 gene coupled with the predicted protein structure of its gene product suggests that the 10 gene product is involved in a signal transduction event important to the differentiation of mature TH cells.
  • the map shown in FIG. 11 indicates that the chromosomal location of the murine 10 gene has been identified.
  • the 10 gene locus is located on Chromosome 12, is closely linked to a class of genes encoding T cell autoantigens, and additionally, maps near the Ig heavy chain gene locus.
  • the nucleotide sequence (SEQ ID NO: 4) of a representative band 57 clone is depicted in FIG. 12.
  • the gene corresponding to band 57 is the 57 gene.
  • the 57 gene appears to be a novel gene sequence in that it does not appear within the published databases. No homology to known peptide domains has, thus far, been identified.
  • the genes 105, 106 and 200 are each expressed at a higher level within the THl cell subpopulation, as revealed by the THl differential appearance of amplified bands 105, 106 and 200.
  • Nucleotide sequences contained within bands 105 and 106 are depicted in FIGS. 13 (SEQ ID NO: 5) and 14 (SEQ ID NO: 6), respectively.
  • the sequence of the murine 200 gene is depicted in FIG. 17 (SEQ ID NO: 8). None of these sequences appear within published databases. Given the THl-specific expression pattern each of these sequences exhibits, the genes and their gene products can potentially be used as treatments for THl-related disorders, as diagnostics for such disorders, and/or as part of methods for the identification of compounds capable of ameliorating THl-related disorders.
  • the 161 gene appears to be TH cell subset specific. That is, 161 gene expression has been observed in either THl cells or in TH2 cells, but its expression has never been observed, simultaneously, in both THl and TH2 cell subpopulations. The details of the 161 gene differential expression pattern are currently being elucidated. It is possible that 161 gene expression is indicative of the presence of yet another TH cell subpopulation, in addition to THl, TH2 and TH0 cell subpopulations.
  • FIG. 15 presents the band 161 nucleotide sequence. While the 161 gene appears to be a novel sequence, it bears a distinct level of similarity to a set of gene sequences (SEQ ID NOS: 13-17) in published databases, as shown in FIG. 16. Interestingly, the genes within this group each contain alpha-interferon responsive promoters.
  • Band 200 was utilized as a probe to identify and isolate murine 200 gene cDNA clones, including clones designated 5 200-P, 200-AF and 200-O, which have been deposited with the NRRL, as summarized in Section 10, below.
  • the cDNA clones were characterized, yielding the full length nucleotide sequence (SEQ ID NO: 8) of the murine 200 gene coding region, as shown in FIG. 17.
  • FIG. 17 also depicts the murine 200 0 gene product derived amino acid sequence (SEQ ID NO: 10).
  • FIG. 18 The results of a murine 200 gene mRNA Northern blot analysis are shown in FIG. 18. The data depicted in FIG. 18 demonstrates, first, that the 200 gene produces a transcript of approximately 1.2 kb in length, and, second, illustrates 0 the THl specificity of 200 gene expression
  • RNA samples were isolated from either unstimulated cells (-) or cells which had been stimulated for 6 hours with plate-bound 5 anti-CD3 antibody (+) .
  • the samples were probed with 200 gene sequences, and, as shown in FIG. 18, RNA from both stimulated and unstimulated THl cells contained 200 gene mRNA, while none of the samples obtained from TH2 cells contained 200 gene mRNA. It should also be noted that 200 gene expression 0 was higher in each of the stimulated THl cells relative to the corresponding unstimulated THl cells.
  • the 54 gene is expressed in a THl-restricted manner.
  • the 54 gene was identified via T cell paradigm searches in which the expression pattern of a 5 THl cell clone, ⁇ E7 , was compared to that of a TH2 cell clone, D10.G4.
  • the initial differential expression analysis was performed using differential display techniques, as described, above, in Section 6.1.
  • the THl-restricted pattern of the 54 gene expression was corroborated through Northern analysis of RNA isolated from THl cell lines (AE7, Dl.l, Dorris) and TH2 cell lines (D10.G4, DAX, CDC25) , as shown in FIG. 19.
  • the TH1/TH2 Northern blot data depicted in FIG. 19 additionally illustrates 54 gene expression within cell clones either stimulated or unstimulated with anti-CD3 antibodies, and demonstrates that 54 gene expression goes down within stimulated THl cells.
  • RNA isolated from AE7 clones was isolated from unstimulated AE7 clones as well as from AE7 clones which had been stimulated with anti-CD3 antibodies for varying lengths of time, as noted in FIG. 20. As illustrated in FIG. 20, 54 gene expression decreased slightly by 2-6 hours after stimulation and had not again achieved pre-stimulation levels within 48 hours after stimulation.
  • a 54 gene expression analysis of cell lines representing a variety of T cells, B cells and monocytic/macrophage cell lines was performed which failed to detect 54 gene expression in non-THl cells, demonstrating that 54 gene expression is highly restricted to THl-like cells.
  • a Northern analysis of 54 gene expression within tissues (FIG. 21) , also demonstrated an expression profile consistent with that of a THl cell-restricted expression profile. Namely, as shown in FIG. 21, most organs failed to express the 54 gene, while the highest level of 54 gene expression was seen in lymph node tissue, and lowest detectable level of expression was seen in spleen, testis and uterus.
  • FIG. 22 shows the entire 54 gene coding sequence (SEQ ID NO: 11).
  • the derived amino acid sequence of the 54 gene product is also shown in FIG. 22 (SEQ ID NO:12).
  • the 54 gene appears to encode a novel cysteine protease.
  • Cysteine proteases are enzymes which contribute to intracellular protein degradation and appear to play a role in tissue degradation. It is possible, therefore, that the inhibition of 54 gene expression and/or 54 gene product activity in immune disorders involving THl- like cells may serve to minimize any tissue damage.
  • the 54 gene sequence exhibits the three thiol protease domains typical of active cysteine protease enzymes. These domains include a CYS daomain at approximately amino acid residue 145 to 156 (active site: C, position 151) , a HIS domain at approximately amino acid residue 287 to 297 (active site: H, position 289) , and an ASN domain at approximately amino acid residue 321 to 340 (active site N, position 326) .
  • the typical CYS domain is broken by a K residue at position 149 (this position is usually G or E) , perhaps indicating that the 54 gene product cysteine protease is very substrate-specific.
  • FIG. 23 depicts the 54 gene product amino acid sequence and points out some of its potential cysteine protease-like features.
  • the 54 gene product has an amino terminal end which resembles a cysteine protease preproenzyme region, which is cleaved away upon formation of the active cysteine protease.
  • the boxed region, from amino acid residue 56 to 75 represents an "ERFNIN" region which has previously been noted as a feature of several cysteine proteases (Ishidoh, K. et al., 1987) FEBS Lett. 226:33-37).
  • the circled amino acid residues within the boxed region represent conserved amino acid residues.
  • the individual boxed amino acid residues represent residues that, based on homology, are thought to lie within the active site of the enzyme .
  • Murine 200 gene probe An approximately 800 bp EcoRI insert containing about 90% of the murine 200 gene cDNA (femt200) ORF was gel purified, 32 P labelled, and used to 5 probe the ⁇ gtll human lymphocyte cDNA library described below.
  • the approximately 500 bp insert of the human 200 gene feht200a cDNA clone was 32 P labelled and used to probe the 0 human fetal spleen cDNA library described below.
  • a human fetal spleen cDNA library constructed using the Stratagene Uni-Zap cloning System was screened using the human feht200a gene probe described above. Approximately 10 6 plaques were hybridized in duplicate at 65°C in Church's buffer overnight. The filters were then washed for 30 min at 5 65°C in 0.1XSSC,0.1% SDS and exposed to film. Positives were confirmed by secondary screening under the same conditions. Subcloning/Sequencing procedures:
  • DNA from the positive clones obtained from the ⁇ gtll cDNA library was generated by a plate lysis method.
  • the purified DNA was digested to obtain cDNA inserts which were subcloned into the pBluescript plasmid (Stratagene) .
  • DNA sequencing was performed according to standard techniques.
  • Northern blot analysis of human gene 200 expression Northern blots were carried out as described in Section 6.1, above. 15 ⁇ g of total RNA from a variety of human organs were analyzed (Clontech, CA) . The 32 P labelled probe utilized was the feht200a clone, described above, which contains the 5' ORF of human gene 200.
  • an 800 bp EcoRI insert containing approximately 90% of the murine 200 gene cDNA (femt200) ORF was gel purified, 32 P labelled, and used to probe a ⁇ gtll human lymphocyte cDNA library. Approximately 10 6 plaques were screened in duplicate, as described in Section 9.1, above. One positive plaque was obtained and rescreened under the same conditions. Once pure, this clone was used to generate lambda DNA by a plate lysis method, and the lambda DNA was digested to obtain a 500 bp insert (feht 200a) which, upon sequencing, was found to be a human homologue of the murine 200 gene.
  • a human 200 gene probe was used to screen a human fetal spleen cDNA library, as described in Section 9.1., above. Three positive clones were obtained, two of which were positive upon secondary screening under the same conditions. The two positive clones were subcloned and their cDNA inserts were sequenced. These two clones labelled feht200b and feht200c were approximately 1.56 kb and 2.0 kb in length, respectively with feht200c containing the entire coding sequence. Clone feht200c was deposited with the ATCC, as described, below, in Section 12.
  • the nucleotide sequence containing the complete human 200 gene open reading frame is depicted in FIG. 24 (SEQ ID NO: 37) .
  • the derived amino acid sequence of the human 200 gene product is also depicted in FIG. 24 (SEQ ID NO: 24) .
  • the 301 amino acid residue sequence of the human 200 gene product reveals that it is a cell surface receptor exhibiting distinct domains, including a signal sequence from amino acid residue 1 to approximately 20, an extracellular domain from approximately amino acid residue 21 to 200, a transmembrane domain from approximately amino acid residue 201-224 and a cytoplasmic domain from approximately amino acid 225 to 301.
  • the extracellular domain contains an Ig type variable set domain from approximately amino acid residue 30 to approximately amino acid residue 128, thus placing the 200 gene product within the Ig receptor superfamily.
  • a Northern analysis of the tissue distribution of 200 gene transcripts was performed. 15 ⁇ g RNA from brain, kidney, liver, lung, muscle, prostate, spleen, thymus and trachea were isolated and analyzed for human 200 gene expression. This analysis revealed human 200 gene transcripts of approximately 2.2 kb, in tissues including brain, lung, trachea, spleen and thymus.
  • the human 200 gene corresponding to the human analog of the murine 200 gene, has been successfully cloned and characterized, as described herein. As revealed by its amino acid sequence, the human 200 gene product is a receptor of the Ig superfamily class of molecules. 10. EXAMPLE: CONSTRUCTION AND EXPRESSION OF IgGl FUSION
  • Reverse oligo 5 ' -TTA-ATT-TGG-ATC-CCC-AGT-TCT-GAT-CGT-TTC- TCC-AGA-GTC-3 ' (SEQ ID NO: 26). 0
  • the oligonucleotide primers also introduce Xhol and BamHI restriction sites at the 5' and 3' ends of the PCR products, respectively, to facilitate the subsequent insertion into IgGl expression vectors (pCD5-CD44-IgGl; see Aruffo, A. et 5 al., 1991, Cell 61:1303-1313).
  • the pCD5-CD44-IgGl vector encodes a protein containing a CD5 signal sequence, a CD44 extracellular domain and a human IgGl heavy chain Fc region.
  • the CD5 and CD44 portions of pCD5-CD44-IgGl were Q replaced with sequences encoding marine 200 gene product signal sequence and extracellular domain.
  • the PCR reactions consisted of 25 cycles amplification at an annealing temperature of 60°C. VentTM thermostable DNA polymerase (New England BioLabs, Inc.; Beverly, 5 Massachusetts) was used in the amplification.
  • the PCR product (approximately 600 bp) was digested with Xhol and BamHI and inserted into pCD5-CD44-IgGl previously digested with Xhol and BamHI to remove the sequences encoding the CD5 signal sequence and the CD44 ectodomain.
  • Forward oligo 5 ' -AAA-TTT-ATT-CTC-GAG-CGC-TAA-CAG-AGG-TGT-CC- 3' (SEQ ID NO: 27) ;
  • Reverse oligo 5 ' -TTA-ATT-TGG-ATC-CCC-TCT-GAT-GGT-TGC-TCC-
  • AGA-GTC-CCG-3 ' (SEQ ID NO: 28).
  • the 103 gene product extracellular domain sequence of the Ig-fusion protein consisted of 103 gene product amino acid residues 27-342 (i.e., the 103 gene product portion ending with amino acid sequence Ile-Val-Ala- Gly-Cys-Ser) .
  • the fragment encoding the 103 gene product extracellular domain was amplified by PCR using synthetic oligonucleotides complementary to the sequences flanking the 103 gene region that would produce the 103 gene product containing amino acid residues 27-342.
  • the oligonucleotides were designed to allow creation of a Kpnl site at the 5' end and a BamHI site at the 3 ' end of each amplified 103 gene fragment to facilitate subsequent insertion into pCD5-CD44-IgGl.
  • the 5' oligonucleotide was as follows: 5'-CCGCGGGTACCAGTAAATCGTCCTGGGGTGG-3 ' (SEQ ID NO: 29).
  • the 3 'oligonucleotide was as follows: 5'-
  • AAATAAAGGATCCCTACATCCAGCAACTATGTAGTA-3 ' (SEQ ID NO: 30). PCR reaction conditions consisted of 15 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, using Vent DNA polymerase (New England Biolabs, Beverly, MA) and 103L gene as template.
  • 103 PCR products were digested with Kpnl and BamHI, and ligated to Kpnl-BamHI sites of CD5-IgGl vector, thus replacing the CD44 sequences with the 103 gene sequences.
  • the resulting plasmid encoding a fusion protein containing CD5-signal sequence, murine 103-extracellular domain and human-IgGl heavy chain Fc region, was transfected into COS cells using LipofectAMINETM (GIBCOBRL, MD) following manufacturer's suggest.
  • O.l ⁇ g plasmid DNA and 140 ⁇ l LipofectAMINETM were used for transfection of the cells of a 150mm plate. Twenty-four hours after transfection, medium was replaced with 10% Ultra-low IgG Fetal Bovine Serum (GIBCOBRL, MD) /DMEM (BioWHITTAKER, Maryland), and the transfected cells were allowed to grow for 4-5 days continuously.
  • Metabolic labelling of recombinant fusion proteins 36 hours after transient transfection of COS-7 cultures, cells were rinsed with replacement growth medium [DMEM methionine and cysteine depleted (ICN, Inc. , CA) ] . After rinsing, 150 ⁇ CI/ml medium of a mixture of 35 S-cysteine and 35 S-methionine (Express 35 s 35 STM , Dupont, MA) was added to the replacement medium and the cells were cultured overnight.
  • replacement growth medium [DMEM methionine and cysteine depleted (ICN, Inc. , CA) ] . After rinsing, 150 ⁇ CI/ml medium of a mixture of 35 S-cysteine and 35 S-methionine (Express 35 s 35 STM , Dupont, MA) was added to the replacement medium and the cells were cultured overnight.
  • hlgGl fusion proteins were generated by LipfectAMINETM (Gibco, Inc., MD) -mediated transient transfection of COS-7 cells according to manufacturer's suggestion for 200 gene- hlgGl fusion proteins, 1 ml of day 5 supernatant was mixed with 20 ⁇ l of Protein A Trisacryl bead (Pierce, Inc., IL) in the presence of 20mM HEPES (pH 7.0) overnight at 4°C with constant agitation. Beads were then washed 3X with PBS prior to the addition to loading buffer.
  • Beads were mixed with either reducing or non-reducing loading buffers (described in, Molecular Cloning, Sambrook, Fritsch, and Maniatis, 2nd edition, 1989, with the exception that DTT was replaced with 2.5% ⁇ -mercaptoethanol) .
  • the construction and expression of recombinant IgG fusion proteins is described herein. Specifically, 200 gene product-IgGl and 103 gene product-IgGl fusion proteins are described.
  • the murine and human 200 gene product-IgGl fusion protein contains a 200 gene product signal sequence and extracellular domain fusion to a human IgGl heavy chain Fc region.
  • the 103 gene product-IgGl fusion protein contains a CD5 signal sequence and 103 gene product extracellular domain fused to a human IgGl heavy chain Fc region.
  • the 200 gene-IgGl fusion protein undergoes oligomerization, as expected, given the human IgGl heavy chain peptide sequence present in the fusion protein.
  • the size i.e. , larger than 5 expected from the amino acid sequence alone
  • appearance of the fusion proteins as they migrate through the gels i.e. , diffuse, rather than tight bands
  • Described herein is the production and characterization of transgenic mice overexpressing either murine 200 gene product or murine 103 gene product. 5
  • a PCR product of the entire 200 gene sequence was used to replace the IL-10 gene in the pCIL-10 plasmid, whose _ construction is described below.
  • the pCIL-10 plasmid contained a 5.5 kb BamHI-Xjbal genomic fragment, within which human CD2 enhancer was included (Greaves et al., 1989, Cell 56(6) :979-86) .
  • a 0.5 kb Xbal-Smal fragment containing human immunoglobulin heavy 5 chain promoter, P ⁇ (Danner and Leder, Proc. Natl. Acad. Sci. USA, 1985, 82 . : 8658-8662) was ligated to the 3 ' -end of the CD2 fragment.
  • a 0.8 kb PCR product of the entire murine 200 gene coding sequence was obtained through 25 cycle-reaction using the murine 200 gene cDNA 200-AF as template and 5 oligonucleotides primers with compatible restriction sites Spel at the 5 ' -end and BamRI at 3 '-end.
  • the 5 '-oligo Utilized was 5 ' -GCG CAA TTG ACT AGT GAC CCA CGC GTC CGG ATT TC-3 ' (SEQ ID NO: 31) and the 3 '-oligo, 5 ' -GAC GCG GAT CCT CAG GAT GGC TGC TGG CTG-3 ' (SEQ ID NO: 32).
  • Transformants were grown in LB broth containing 0.1 ⁇ g/ml ampicillin and the DNA were extracted by Qiagene Plasmid Maxi Kit (Qiagene, CA) . Restriction analysis was performed for confirmation, and the final construct was sequenced to eliminate any possible PCR introduced mutations.
  • a plasmid designated p200Tr3 was selected from production of transgenic mice.
  • This final construct contained an approximately 5.5 kb genomic fragment containing the human CD2 enhancer joined to a 0.5 kb fragment of the human IgM promoter immediately upstream of the murine 200 gene coding sequence.
  • a region containing the 3 ' untranslated sequence of the human growth hormone gene was positioned immediately downstream of the murine 200 gene ORF and contained a polyA splice site.
  • a PCR product of the entire 103 gene sequence was used to replace the IL-10 gene in the pCIL-10 plasmid.
  • the pCIL-10 plasmid was as described in this Section, above.
  • RNAzoleTM TEL-TEST, Inc. , TX
  • the PCR product was digested by Spel and BamHI (New England Biolabs) and ligated into the Spel-BamHI sites of pBSKIIGH vector, containing the human growth hormone fragment from pCIL-10 subcloned into the BamHI-XhoI site of pBSKII (Stratagene) , which was named pBS-103L-GH.
  • the pCIL-10 fragment containing human CD2 enhancer and P ⁇ promoter was then ligated immediately upstream of the 103L gene of pBS-103L-GH.
  • MaxEfficient E. coli DH5 competent cells (GIBCO BRL, MD) were used for transformation following manufacturer's suggestion.
  • the transformants were grown in LB broth containing 0.1 ⁇ g/ml ampicillin and DNA were extracted by Qiagene Plasmid Maxi Kit (Qiagene, CA) . Restriction analysis was performed for confirmation, and the construct was sequenced to eliminate any possible PCR introduced mutations. A plasmid designated pCD2-103L-GH was selected for production of transgenic mice. Production of Transgenic Mice
  • C3H/HEJ and FVB/NJ mice were obtained from the Jackson Laboratory (Bar Harbor, ME) .
  • Females aged 3-4 weeks were induced to ovulate by intraperitoneal injection of pregnant mare's serum (PMS) between 10 a.m. to 2 p.m., followed 46 hours later by intraperitoneal injection of human chorionic gonadotropin (hCG) .
  • PMS pregnant mare's serum
  • hCG human chorionic gonadotropin
  • the females were housed overnight with males of the same strain.
  • the following morning females were examined for the presence of a copulation plug and embryos were isolated from those females with plugs, essentially as described in Manipulating the Mouse Embryo (Hogan et al., eds. , Cold Spring Harbor Laboratory Press, 1994) .
  • DNA for embryo microinjection was prepared by digesting of p200Tr3 and pCD2-103L-GHl with WotI and Xhol followed by gel electrophoresis.
  • the 9 kb and 10 kb fragments, respectively, were electrophorese onto an NA-45 membrane (Schleicher and Schuell) by cutting a slit into the gel immediately in front of the desired band, inserting the NA-45 membrane and continuing electrophoresis until the DNA band has been transferred to the membrane.
  • the DNA was eluted from the membrane by incubation with 0.4 ml of 1M NaCl/0.05M arginine-free base at 65-70°C for several hours in a microfuge tube.
  • the eluted DNA was extracted with phenol/chloroform and chloroform, ethanol precipitated and dissolved in 200 ⁇ l of 5 mM Tris, pH 7.5/0.1 mM EDTA.
  • the DNA was then re-precipitated with ethanol and re-dissolved in 40 ⁇ l of 10 mM Tris, pH 7.5/0.1 mM EDTA.
  • the DNA Prior to microinjection, the DNA was diluted to 1-2 ⁇ /ml in 10 mM Tris, pH 7.5/0.1 mM EDTA.
  • radiolabelled probe containing approximately 500 base pairs of the human IgM promoter was produced using the Random Primed DNA Labelling Kit (Boehringer Mannheim) .
  • the 500 bp Xba-l/Spe-1 fragment of human IgM heavy chain promoter was used as probe.
  • Hybridization was carried out using standard hybridization procedures using Rapid-Hyb (Amersham) hybridization solution. 1 x 10 6 cpm per ml of hybridization solution was incubated at 65°C overnight. Membranes were washed twice in 0.5X SSC 0.1% SDS at 65°C for 30 minutes and were exposed by autoradiography . Transgenic animals were detected by the presence of an approximately 7.0 kb BamHI fragment which hybri ⁇ izes to a probe containing the 0.5 kb P ⁇ fragment.
  • PCR fragment For 103 gene transgenic animals, a 32 P-radiolabelled PCR fragment of the pCD2-103L-GH construct described above was utilized. The PCR fragment was generated using the following primers: 5' oligo: 5 ' -GTA-AAT-CGT-CCT-GGG-GTC-TGG-3 ' (SEQ ID NO:35; 3' oligo: 5 ' -CCT-TCT-GAT-AAC-ACA-AGC-ATA-AAT-C-3 * (SEQ ID NO: 36).
  • PCR reactions conditions were as follows: 20 cycles of 30 seconds at 94°C, 30 seconds at 60°C and 30 seconds at 72°, using VentTM DNA polymerase (New England Biolabs, Beverly MA) .
  • VentTM DNA polymerase New England Biolabs, Beverly MA
  • the resulting probe hybridized to an endogenous 2.4 kb band and a 0.85 kb transgenic-specific band.
  • transgenic mice With respect to the 200 transgenic animals, four lines of transgenic mice were created in the C3H inbred strain of mice. One of these lines was examined for expression of the 200 transgene. As expected, 200 transgene transcripts were detected in the thymus, spleen and lymph nodes, consistent with a predominantly T-cell restricted pattern. At approximately 6 to 7 months of age, three of the founder animals, upon visual examination, appeared sick. One of these founders, designated 130-1.2, was sacrificed at approximately 6 months of age. At the time the sacrifice, it was expected that at the female would not have lived significantly longer. Upon dissection of 130-1.2, it was clear that the spleen and one of the kidneys were grossly abnormal.
  • the spleen was approximately ten-fold normal size and appeared to be filled with pale appearing cells.
  • the splenocyte populations were examined by flow cytometry, and it was determined that the predominant cell population was positive for MAC-1 (a macrophage/granulocyte cell surface marker) expression. These cells also had high side scatter profiles. Spleen sections from this animal were stained with hematoxylin and eosin and viewed by light microscopy. These data suggest that the abnormal cell population was composed of polymorphonuclear neutrophils. The abnormal kidney also appeared to be infiltrated by these same cells. One of the offspring of 130-1.2 died at approximately 6 months of age while giving birth to her second litter.
  • transgene is modulating some aspect of the immune system.
  • the line may suffer from an immunodeficiency and is, therefore, susceptible to infection by normally innocuous organisms present in the environment (bacteria, etc.). It is possible, therefore, that this gene product normally functions in some aspect of the immune effector response or in the proper regulation of the immune system.
  • Two hundred transgenic mouse founder lines generated in the FvB inbred strain exhibited no outward symptoms of illness as they approached 6 months of age.
  • the Example herein presents in vivo data demonstrating that the 103 gene product regulates TH2 effector cell responses.
  • a monoclonal antibody (3E10 mAb) has been generated against the 1C3 gene product, and its effect in an adoptive transfer model of THl and TH2 immune responses was investigated.
  • the effect of a 103 gene product fused to an Ig tail (103/Ig fusion) has also been studied in the adoptive transfer model.
  • the anti 103 gene product mAb abrogated the production of IL-4, IL-5, IL-6 and IL-13, TH2 mediated lung inflammation and the associated airway hyperresponsiveness. Likewise, the 103/Ig fusion results in a decrease in eosinophil infiltration into and inflammation of lung airways. In contrast, the 103 gene product mAb failed to inhibit TH1- mediated lung pathology and IFN- ⁇ secretion. These results, therefore, provide in vivo animal data indicating that the 103 gene product provides a critical signal to TH2 effector cells and can be utilized as a novel target for the selective suppression of TH2 immune responses.
  • CD3/TCR Crosslinking Mice expressing the transgene for the DOll.lO ⁇ -TCR, which recognizes residues 323-339 of chicken ovalbumin (OVA) in association with I-A d (Murphy, K.M. , et al., 1990, Science 250: 1720-1723) were utilized.
  • DOll.lO TCR-transgenic CD4 + T cells were cultured in complete RPMI 1640 with OVA 323 _ 339 (1 ⁇ M) and mitomycin C-treated splenocytes.
  • recombinant murine 11-12 (lOng/ml) and neutralizing anti-IL-4 mAb (11B11, 40 ⁇ g/ml, R&D Systems) were added and for TH2 development recombinant murine IL-4 (lOng/ml) and neutralizing polyclonal anti-murine IL-12 (TOSH-2, 3 ⁇ g/ml, Endogen, Cambridge, MA) were used.
  • Cultures were maintained for 48 hours and 5 days after stimulation, after which time cells were harvested and purified over ficoll. 1 x 10 7 cells were washed and RNA extracted as described below. The remainder of the cells were stimulated on plate bound anti-CD3 in the presence of h IL-2 (Endogen) for 48 hrs.
  • Rat monoclonal antibodies including the 3E10 MAb, were generated against the extracellular domain of the mouse 103 gene product.
  • a DNA sequence containing the extracellular domain of 103 gene product was PCR-amplified and cloned into a vector containing the CD5 signal sequence and the human IgGl constant region.
  • COS cells were transiently transfected using lipofectamine tm (GIBCO) protocol according to manufacturer's instructions. Cells were cultured in Ultra-LowTM Ig fetal bovine serum
  • Lou/M rats were then immunized by subcutaneous injection of 0.5 mg purified recombinant 103 gene product protein. Rats were boosted twice via intraperitoneal injections at 2 week intervals with approximately 300 ⁇ g purified protein. Animals were analyzed for reactivity to the fusion protein by FACS and ELISA approximately 10 days after the last boost. Four weeks later, positive reacting animals were boosted once more and sacrificed 3 days later.
  • Splenocytes were fused with SP/2 myeloma cells and resulting clones were screened and selected to be specific for the 103 gene product on the basis of their reactivity against 103 gene product Ig, but not CD44-Ig, and their ability to stain 103 gene product COS transfectants, but not control transfectants.
  • Pre-immune serum from non-immunized Lou/M rats was used as negative controls.
  • the 3E10mAb was labeled with digoxigenin and the number of 103-positive cells were detected by anti-digoxigenin Fab fragments (Boehringer Mannheim) conjugated to Cy5.
  • CD4 positive, L-selectin negative cells were isolated using high gradient magnetic cell separation system MACS (Milteny; Biotec, Berg-Gladbach) .
  • Expression of 103 was analyzed on a fluorescence-activated cell sorter (FACS)- calibur (Becton-Dickinson) five to seven days after restimulation with OVA peptide under the indicated polarizing conditions .
  • FACS fluorescence-activated cell sorter
  • Becton-Dickinson Becton-Dickinson
  • TH2 effector cells were activated with platebound CD3 (l ⁇ g/ml, 2C11) and CD28 (37.51, 4 ⁇ g/ml) , Pharmingen, San Diego) and 3E110 (20 ⁇ g/ml) for 48 hours.
  • IL-4 and IL-5 levels were measured in the supernatent by Elisa.
  • RNA isolation Total cellular RNA for RT-PCR analysis was extracted from cells using the Rneasy Total RNA kit (Qiagen; Chatsworth, CA) . Poly A+ RNA (for Northern analysis) was isolated from activated cells using FastTrack mRNA Kit (Invitrogen Corp.; San Diego, CA) .
  • RNA were loaded per lane for the Northern blot analysis.
  • the 103 gene probe was a 409 bp Rsal fragment from the 103 gene cDNA (position 1252-1661 based on the published sequence for Genbank accession number D13695) .
  • IL-4 and beta-actin probes were purchased from Clontech, Inc. , Palo Alto, CA.
  • IFN- ⁇ probe consisted of a 344bp fragment of murine IFN- ⁇ covering the region from position 532-876 (genbank accession number M28621) .
  • Northern blot analysis was carried out according to standard techniques.
  • RT-PCR First strand cDNA was synthesized from equal amounts of RNA using the Superscript Preamplification System (Life Technologies; Gaithersburg MD) . PCR was performed using 25 ng of first-strand cDNA. The following gene- specific primers were used for PCR amplification: Gene 103: 5' ACGGAGGGCAGTAAATC and 5' CAGCCAAGAAGTGAGAGC; IFN-gamma 5' TGTTGCCGGAATCCAGCCTCAG and 5' GTCCCCCACCCCCAGATACAACC .
  • G3PDH glyceraldehyde 3- Phosphate Dehydrogenase
  • IL-4 glyceraldehyde 3- Phosphate Dehydrogenase
  • PCR was carried out using the Advantage KlenTaq Polymerase mix (Clontech Laboratories; Palo Alto, CA) according to the provided protocol; annealing temperature 56° C. Samples were removed from the PCR reaction beginning after 15 cycles and then after 5-cycle increments. Reactions using the minimum number of cycles to visualize the gene of interest, were loaded onto 1.5% agarose gels for analysis.
  • TH recipient mice THl and TH2 subsets were generated as described above. Mice were injected with 2 x 10 6 TH2 cells intravenously into recipient BALB/c mice. Twenty four hours later, mice were exposed daily to an aerosol of OVA (50 mg/ml) (Grade V, Sigma, St. Louis) for 20 min for 2 consecutive days. Control mice were either injected with TH2 cells and exposed to an aerosol of PBS or were exposed to OVA in the absence of cell transfer. Mice were sacrificed 24 hrs after the last aeroallergen challenge.
  • OVA 50 mg/ml
  • mice were injected with either 20 ⁇ g or 100 ⁇ g of 3E10 mAb, recombinant 103 gene product-IgG fusion protein, or 100 ⁇ g of rat IgGl (Sigma, St. Louis) as the appropriate isotype control. Twenty fours after the last challenge, the trachea was cannulated and a bronchoalveolar lavage performed with 4 x 0.3 ml aliquots of PBS (Gonazlo, J.A. , et al., 1996, Immunity 4:1-14). Cytokine levels in the lavage fluid were measured by ELISA (PharMingen, San Diego) .
  • TH clones AE7 (THl) , Dorris (THl), DAX (TH2) and D10.G4 (TH2) clones were analyzed for the expression of gene 103 protein using fluorescence activated cell sorting (FACS) . Cells were stimulated with appropriate antigen and cultured for approximately 3 days prior to analysis. Pre-im une serum was prepared for unimmunized Lou/M rats.
  • FACS fluorescence activated cell sorting
  • Cell Preparation and polarization Spleens from DOll.ll OVA a ⁇ TCR mice were removed and CD4+ T cells were purified by negative selection. Cells were plated at a density of 1 x 10 6 / ⁇ r ⁇ l in 75 mm 2 flasks and stimulated with 10 ⁇ g/ml OVA peptide and mitomycin C treated splenocytes at a ratio of 1:1 CD4 : APC. Cells were cultured in the presence of IL-4 (20 ng/ml) and anti-IL-12 (3 ⁇ g/ml) for TH2 polarization, or IL- 12 (20 ng/ml) and anti-IL-4 (40 ⁇ g/ml) for THl polarization.
  • IL-4 (20 ng/ml) and anti-IL-12 (3 ⁇ g/ml) for TH2 polarization
  • IL- 12 (20 ng/ml) and anti-IL-4 (40 ⁇ g/ml) for THl polarization.
  • Adoptive transfer model 2 x 10 6 cells were injected intravenously via the tail vein into recipient transgenic mice. Twenty four hours later, mice were exposed daily to an aerosol of OVA (50 mg/ml) antigen (Grade V, Sigma, St. Louis) for 20 minutes. Control mice were exposed to an aerosol of PBS alone. Mice were sacrificed on days 3, 5 and 7. In separate experiments, mice received 20 ⁇ g/mouse i.v. of either 3E10 MAb or the 103 Ig fusion protein. Control mice were injected with 20 ⁇ g of either rat or human Ig as the appropriate isotype control. This procedure was repeated for two consecutive days.
  • mice were anaesthetized with 0.3 ml of 14% urethane i.p. and the trachea cannulated.
  • a bronchoalveolar lavage (BAL) was performed by injecting 0.3 ml of PBS into the lungs. The fluid was then withdrawn and stored on ice. This procedure was repeated a total of 4 times. The cell suspension was then centrifuged (5 mins, 1500 rpm, 4° C) and the supernatant removed and frozen at -20° C. The cell pellet was then resuspended in 1 ml of PBS and total cell counts were obtained. Cytospin preparations were then prepared and stained with Diff-Quik (Baxter Corporation) . A total of 200 cells were then counted differentially using standard morphological criteria. Cytokine levels were measured in the BAL fluid by ELISA (Pharmingen, San Diego) .
  • mice Male BALB/c mice (15-20 g) were immunized intraperitoneally with 7.5 ⁇ g of OVA and 1.5 mg A1(0H)3 in saline on Day 0 and Day 7. On day 14 and Day 21 the mice were challenged with aerosolized OVA (10 mg/ml) for 1 hours. Control mice were challenged with PBS instead of OVA. One hour prior to each allergen sensitization and challenge, the mice were injected with 100 ⁇ g of 3E10 Mab or 100 ⁇ g of rat IgGl (Sigma, St. Louis) . Twenty-four hours following the second allergen challenge a BAL was performed and IL-5 levels in the BAL fluid determined. Serum OVA specific IgE was determined by specific ELISA.
  • Airway responsiveness was measured in TH2 recipient mice, 24 hours after the last aerosol challenge by recording respiratory pressure curves by whole body plethysmography (Hamelmamn, J.E., 1997, Am. J. Respir. Crit. Care Med. 156:766-775); Buxco®, EMKA Technologies, Paris, France) in response to inhaled methacholine (Aldrich-Chemie, Steinhein, Germany) at a concentration of 2.5 to 25 mg/ml for 1 minute. This method allowed measurements of spontaneous breathing in a non- restrained mouse.
  • Airway responsiveness was expressed in enhanced pause (Penh) , a calculated value, which correlates with measurement of airway resistance, impedance and intrapleural pressure in the same mouse.
  • Lung Histology Following the BAL analysis, lungs were inflated with 0.6 ml of a mixture of OCT compound (Tissue- kek®; Miles Inc., Elkhart, IN) and 20% sucrose (Sigma, St. Louis, MI) at a ratio of 1:1. The lungs were then removed, snap-frozen and 8-10 ⁇ m cryosections fixed in methanol at 20°C for 2.5 minutes. Slides were stained with haematoxylin and eosin (Fluka Chemika, Buchs, Switzerland) .
  • In situ Hybridization Recipient Balb/C mice were injected intervenously with 2xl0 6 THl or TH2 cells generated as described above. Twenty-four hours later, mice were exposed to an aerosol of OVA (50 mg/ml; Grade V, Sigma) for 20 minutes for two consecutive days. Mice were sacrificed 24 hours after the last aeroallergen challenge. Lungs were removed and snap frozen for in situ hybridization.
  • OVA 50 mg/ml
  • Grade V Grade V
  • a 35-mer antisense oligonucleotide against 3 ' -UTR 103 gene sequence was synthesized and end-labeled as follows: 100 pmol oligo was incubated for 15 minutes at 37°C with 10 nmol dATP (Promega) , 40 ⁇ mol biotin-dUTP, lx terminal transferase buffer, 5 mM CoCl 2 , 50 units transferase (Boehringer-
  • RESULTS RT-PCR analysis demonstrates that the 103 gene is induced only upon CD3/TCR crosslinking during differentiation of TH0 to TH2 , but not THl effector cells.
  • the RT-PCR analysis was confirmed by Northern analysis. These data corroborate the results presented in the Example of Section 7, above.
  • a monoclonal antibody (3E10 mAb) directed against the extracellular domain of the 103 gene product was prepared and characterized.
  • FIG. 25 Flow cytometry data is presented in FIG. 25 which demonstrates that the 3E10 mAb recognizes and binds to representative clones of the TH2 cell subpopulation (D10.G4; DAX), but not clones of the THl subtype (AE7 ; Dorris).
  • THl serum positive control
  • rat l03 serum positive control
  • this mAb failed to recognize resting or activated CD4+ (L-selectin) , CD8+, B cells or macrophage cells.
  • pretreatment of TH2 recipient mice with 3E10 mAb inhibited the secretion of IL-4, IL-5, IL-6 and IL-13 by greater than 90%.
  • analysis of the cytokine profile in the BAL revealed high levels of IL-4, IL- 5, IL-6, IL-10 and IL-13 in TH2 recipient OVA challenged mice (closed bars) .
  • Pretreatment with 3E10 mAb resulted in a dramatic reduction in IL-4, IL-5, IL-6 and IL-13, but had no effect on IL-10 levels in the BAL (open bars) .
  • OVA challenge of THl recipient mice resulted in high levels of IFN- ⁇ in the BAL fluid (closed bars) that was not inhibited by 3E10 mAb (open bars) .
  • These data show that the 103 gene is differentially expressed in a TH2-specific manner, thereby corroborating the results presented in the Example of Section 7, above.
  • the data demonstrate the feasibility of using antibodies to separate TH2 subpopulation cells away from other cell types, thereby modulating a TH cell subpopulation by changing the number of cells belonging to one TH cell subpopulation relative to that of another TH cell subpopulation.
  • mice which had received TH2 cells and control rat Ig resulted in infiltration of lymphocytes and eosinophilic inflammation of the airways.
  • In vivo administration of 3E10 mAb markedly suppressed the development of eosinophilic inflammation of the airways.
  • eosinophilic inflammation was assessed, first, by histological analysis of the airway tissue. Second, an analysis of the cellular composition of the bronchoalveolar lavage fluid (BAL) was performed (FIGS. 27A-27B) .
  • BAL bronchoalveolar lavage fluid
  • TH2 mediated lung mucosal eosinophilic inflammation is associated with heightened airway responsiveness to non specific stimuli and is a characteristic feature of bronchial asthma (Ohashi, Y. et al., 1992, Am. Resp. Dis. 145: 1469-76) .
  • bronchial asthma Ohashi, Y. et al., 1992, Am. Resp. Dis. 145: 1469-76
  • the degree of airway constriction induced by the methacholine inhalation was assessed using whole body plethysmography.
  • 3E10 mAb treatment was, indeed, demonstrated to attenuate allergen induced heightened airway responsiveness.
  • 3E10 mAb treatment suppressed the development of airway hyperresponsiveness induced by OVA challenge in TH2 recipient mice (FIG. 28) .
  • TH effects of treatment with 3E10 mAb were comparable to those previously reported using anti- IL-5 mAbs Wang, L.M. , 1992, EMBO 11:4899-4908 and anti-B7-2 Abs (Tsuyuki, S. et al., 1997, J. Exp. Med. 185:1671-1679) .
  • the level of interferon gamma was measured, which represents a hallmark of THl cell subpopulation activation, and an increase in its level was detected. This indicates the presence of a relative increase in THl cell subpopulation responses.
  • mice treated with a soluble fusion protein containing the extracellular domain of the 103 gene product fused to an Ig tail (103/Ig fusion) .
  • Administration of the 103/Ig fusion results in significant decrease in hallmark symptoms of asthma.
  • FIG. 29B such administration results in animals that exhibit a decrease in eosinophil infiltration into lung airways (this was assessed by both BAL and histological examination) .
  • administration of the 103/Ig fusion resulted in a 50% attenuation in the degree of eosinophilic inflammation of airways.
  • the inhibition of 103 gene function appears to modulate TH cell subpopulations by decreasing the level and/or activity of TH2 cells while bringing about a relative increase in the level and/or activity of THl cells.
  • TH2 effector cells were activated with plate bound CD3 and CD28. Under conditions where Fc crosslinking occurred, 3E10 mAb augmented IL-4 and IL-5 secretion in the absence of enhanced proliferation (FIG. 30) . In contrast, CD3/CD28 stimulation of THl cells in the presence of plate bound 3E10MAb failed to modify IFN- ⁇ secretion.
  • GATA-3 have been shown to be preferentially expressed in TH2 cells and suggested to play an important role in TH2 differentiation (Zheng, W.-P. & Flavell, R.A. , 1997, Cell 89:587-596).
  • the 103 gene product is induced upon CD3/TCR mediated activation and not during TH2 differentiation from TH0 cells.
  • GATA-3 may be involved in TH differentiation, while the 103 gene product may be more involved during activation of TH2 effector cells.
  • the 103 gene promoter in murine mast cells contains a GATA-3 consensus binding sequence (Gachter, T. et al.,
  • these results provide both in vitro characterization of 103 gene expression and the 103 gene product, as well as in vivo animal data indicating that the 103 gene product provides a critical signal to TH2 effector cells and represents a critical regulatory molecule for both cellular and humoral allergic inflammation. These data indicate that the 103 gene and/or gene product can be utilized as a novel target for the selective suppression of TH2 immune responses .
  • the Example provided herein presents in vivo data that the 200 gene product is involved in the recovery from kidney ischemia injury.
  • a monoclonal antibody is involved in the recovery from kidney ischemia injury.
  • the anti-200 mAb markedly impaired restoration of renal function, as determined by several indicators.
  • the 200 gene product provides a critical function in the resolution of injury following kidney ischemia.
  • the 200 gene and its gene product can be used to aid in the recovery from ischemic injuries such as stroke, heart attack, acute renal failure, and organ transplant.
  • MATERIALS AND METHODS anti-200 mAb Rat monoclonal antibodies (mAbs) were generated against the extracellular domain of the mouse 200 gene product. Recombinant murine 200 gene IgGl (m200Ig) fusion protein was produced and purified as described in Section 10.1 above. Briefly, a DNA sequence containing the extracellular domain of the mouse 200 gene product was PCR- amplified and cloned into a vector containing the CD5 signal sequence and the human IgGl constant region. COS cells were transiently transfected using lipofecta ineTM (GIBCO) according to the manufacturer's instructions.
  • GIBCO lipofecta ineTM
  • mice were analyzed for reactivity to the fusion protein by FACS and ELISA 10 days after the last boost. Four weeks later, a selected animal which demonstrated positive reactivity to the m200Ig fusion protein was boosted once more with 500 mg of m200Ig and sacrificed three days later. Splenocytes from this animal were fused with SP2/0 myeloma cells, and resulting clones were screened and selected to be specific for the murine 200 gene product on the basis of their reactivity to clonal TH2 cell lines (DAX, and D10.G4) and thymocytes from m200 transgenic mice versus non-transgenic mice.
  • DAX clonal TH2 cell lines
  • a single hybridoma termed 96.3.8H7, was identified which selectively reacted to all THl cell lines and m200 transgenic thymocytes, but did not react to TH2 cell lines and non-transgenic thymocytes .
  • Surgical Methods Mice were anesthetized with a single cocktail of Ketamine (200 mg/kg) plux Xylazine (10 mg/kg) injected intraperitoneally . 1 ml of 0.9% dextrose saline was also administered subcutaneously. Core body temperature was maintained at around 37 °C. Using a midline abdominal incision renal arteries and veins were bilaterally occluded for 32 minutes with microaneurysm clamps, during which time the abdomen was closed. After the renal pedicle clamps were removed, the kidneys were observed for an additional five minutes to document color change indicating reflow, and the incision was sutured.
  • Ketamine 200 mg/kg
  • plux Xylazine 10 mg/kg
  • mice were pretreated 24 hours before surgery with 100 ⁇ g/mouse of rat anti-200 monoclonal antibody (200 mAb) .
  • mice were administered equivalent dosages of rat Ig (Rtlg) antibody. Mice were then given antibody at 24 hour intervals following surgical recovery (4 hours post anesthetic) .
  • mice were housed in metabolic cages for 24 hours prior to sacrifice ir order to collect urine for protein analysis as a measure of renal dysfunction.
  • the animals were exsanguinated by cardiac puncture under terminal anesthesia (induced by carbon dioxide in a precharged chamber) and the kidneys were removed. Serum was analysed for urea nitrogen and creatinine by Tufts Vetinary 5 Diagnostic Laboratories.
  • One half of each kidney was frozen in liquid nitrogen for RNA extraction, one quarter was fixed in 10% buffered formalin for histological assessment by Hematoxylin and eosin, and the remaining quarter was frozen for in situ hybridization and immunohistochemical analysis.
  • Kidney Histology Kidneys were processed, sectioned and stained with hematoxylin and eosin according to standard methods. The resulting sections were examined blind and were assigned an arbitrary score based upon the presence or
  • focal interstitial inflammatory infiltrate focal interstitial inflammatory infiltrate
  • diffuse interstitial inflammatory infiltrate diffuse interstitial inflammatory infiltrate
  • glomerular leukocytic infiltrate protein deposits in the tubule
  • protein deposits in the tubule presence of apoptotic bodies in the tubular epithelium, flattened or denuded tubular
  • the 200 gene product is similar to the rat adhesion molecule called KIM-1 (Ichimura et al . , 1998, J . Biol . Chem .
  • a monoclonal antibody (96.3.8H7 mAb) directed against the extracellular domain of the 200 gene product was prepared and characterized, and this antibody was administered to mice
  • Control animals were adminstered equal dosages of rat Ig at the same intervals.
  • a second group of animals underwent sham surgery, described in Section 13.1 above, wherein the animals were not subjected to ischemic kidney injury.
  • Serum creatinine and blood urea nitrogen (BUN) levels were measured in animals sacrificed 24, 48, 72, and 96 hours after kidney reperfusion. These levels are shown in Table 3 below. Creatinine and blood urea nitrogen levels returned to basal levels in untreated (+Rtlg) mice within 72 hours post ischemia. However, mice treated with anti-200 mAb (+a200) maintained elevated levels of both blood urea nitrogen (122.5 mg/dl vs. 34.3 mg/dl) and creatinine (0.73 mg/dl vs. 0.3 mg/dl) .
  • FIG. 31 shows histological cross sections of kidney tisue from untreated (Fig. 31A) and treated (Fig. 31B) rats at 72 hours post reperfusion. Treated mice still exhibited severe tissue damage with flattened tubular epithelial cells, leukocyte infiltration, and tubular cast. The mean histological score was +6 in anti-200 treated mice compared to control mice at 72 hours (Fig. 32) .
  • the example provided herein presents data demonstrating that the 103 gene product is expressed, in mammalian mast cells.
  • Northern analysis showed high levels of 103 gene expression in a human mast cell line.
  • the example also describes the production of monoclonal antibodies which are specific for the human, but not mouse, 103 gene product. FAC staining of the human mast cell line demonstrated binding of these monoclonal antibodies, confirming that the 103 gene product is, indeed, expressed in mast cells.
  • MATERIALS AND METHODS 103 fusion proteins Monoclonal antibodies (mAbs; see below) were generated against the extracellular domain (amino acid residues 18-323) of a human 103 gene product (with a glutamic acid at amino acid residue 78) .
  • the following fusion protein was utilized: a fusion protein that contained, from amino- to carboxy-terminus, a CD5 signal sequence (CD5ss) plus GT residues, the extracellular domain of the 103 gene product described above.
  • a second Fc fusion protein of the human 103 gene product was constructed, according to the techniques described in Section 10.2 above, to bind ELISA plates for screening.
  • the protein contain, from amino- to carboxy- terminus, a T075 signal sequence (T075ss) plus QR residues, amino acid residues 20-323 of the human 103 gene product plus a linker amino acid sequence (Ala-Ala-Ala-Asp-Pro) and a human IgGl constant region.
  • a fusion protein of the mouse 103 gene product was also utilized that contained, from amino- to carboxy-terminus, a CD5 signal sequence plus GT residues, residues 24 (Thr) to 328 (Pro) of the mouse 103 gene product, and human IgGl constant region.
  • 5 A control fusion protein were also utilized which contained unrelated proteins (T001, a che okine, or T075, a tumor necrosis family receptor) fused with a human IgGl constant region.
  • the human IgGl sequence was a in Aruffo et al., 1990, Cell 61:1303.
  • MAbs were generated in Balb/c mice against the extracellular domain (amino acid residues 18-323) of a human 103 gene product (with a glutamic acid at amino acid residue 78) utilizing the above-described fusion protein, coupled with standard techniques (see Section 5.6, above) for mAb generation, selection and purification.
  • Splenocytes from animals which showed positive reactivity to the 103 fusion protein were fused with SP2/0 myeloma cells, and the resulting clones were screened and selected to be specific for the human 103 gene product by ELISA and by Biacore (BIAcore, Inc.; Uppsala, Sweden) as described in Fagerstam et al . , 1992, J . of Chromatography 597:397-410 and in Kretschmann & Raether, 1968, Z . N aturf or s chung Mol . A . 23 : 2135 .
  • Twenty-one clones were shown to specifically bind the human 103 gene product (with either alanine or glutamic acid at amino acid residue 78) but not to a mouse 103-gene product Fc fusion protein or to a control Fc fusion protein.
  • Northern Blots Northern procedures performed in the experiments described in this example were performed as described, above, in Section 6.1.
  • Flow cytometry analysis of mast cell lines Cells were analyzed for the expression of gene 103 protein using fluorescence activated cells sorting (FACS) according to standard methods described in Section 12.1, above using anti- mouse IgFITC secondary antibodies. 14.2. RESULTS Northern blot analysis of multiple cells lines showed high levels of the 103 gene in a human mast cell line. Expression of the 103 gene product in this cell line was verified using monoclonal antibodies raised against an Fc fusion protein of the human 103 gene product, as described in Section 14.1, above.
  • FACS fluorescence activated cells sorting
  • FACS staining of the human mast cell line with the 21 monoclonal antioodies showed staining with 15 of the 21 antibodies compared to isotype controls. Five of these 15 antibodies, identified as 1B4 , 203, 3F7, 3H18 , and 10F7, were selected for further analysis. FAC staining with these antibodies was demonstrated to be specifically blocked with an excess of human 103-Fc fusion protein, however, staining was not blocked with control Fc fusion proteins.
  • the 103 gene product is expressed in a human mast cell line.
  • the 103 gene, its gene product, and compositions derived therefrom may be used not only in the treatment and regulation of TH2 immune disorders such as asthma, but may also be used in the treatment and regulation of mast cell related disorders.
  • mast cell related disorders include, but are not limited to, atherosclerosis (see, e.g. , Metzler and Xu, 1997, Int . Arch . Allergy Immunol . 114:10-14) and myocardial ischemia/reperfusion (see, e. ⁇ r. , Frangogiannis et al . , 1998, Circulation 98:699-710).
  • accession numbers The following microorganisms were deposited with the American Type Culture Collection (ATCC) , 10801 University Boulevard, Manassas, Virginia, on December 12, 1995 and assigned the indicated accession numbers:

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Abstract

L'invention concerne des procédés et des compositions pour le traitement et le diagnostic de troubles de l'immunité, notamment pour les troubles liés aux lymphocytes T et aussi pour le traitement de pathologies et troubles associés à des basophiles/mastocytes, de troubles et de lésions ischémiques, comprenant les troubles et lésions rénaux ischémiques. Les gènes, par exemple, exprimés de manière différente dans et parmi les lymphocytes T et les sous-populations de cellules TH2, sont identifiés. Les gènes sont aussi identifiés par la capacité de leurs produits génétiques à interagir avec les produits génétiques intervenant dans la différentiation, l'entretien et la fonction effectrice de ces lymphocytes TH et de ces sous-populations de lymphocytes TH. Les gènes identifiés peuvent contribuer au diagnostic ou servent de cibles pour l'intervention thérapeutique. Dans ce sens, l'invention concerne des procédés d'identification et d'utilisation thérapeutique de composés comme traitement des troubles de l'immunité, plus particulièrement des troubles associés aux sous-populations de lymphocytes TH. L'invention concerne en outre des procédés d'évaluation diagnostique et de pronostic des troubles associés aux sous-populations de lymphocytes TH, d'identification des sujets enclins à être affectés par de telles pathologies, de suivi des patients sous évaluation clinique pour le traitement de ces troubles et de surveillance de l'efficacité des composés utilisés dans les essais cliniques. Font finalement l'objet de cette invention des procédés pour le traitement de symptômes associés à des pathologies ou troubles liés aux basophiles/mastocytes, aux troubles et aux lésions ischémiques utilisant les gènes, les produits génétiques et les anticorps.
PCT/US2000/014986 1999-06-02 2000-05-31 Compositions et procede pour le traitement et le diagnostic de troubles de l'immunite Ceased WO2000073498A1 (fr)

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US09/324,986 1999-06-02

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US20090041752A1 (en) 2009-02-12

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